Ii peptide therapeutics to enhance antigen presentation

ABSTRACT

Disclosed is a class of compounds referred to herein as effector compounds. Effector compounds are useful in connection with the modulation of an immune response. Modulation refers to the ability of the effector compounds of the present invention to either enhance (antigen supercharging) or inhibit (immunosuppressant activities) antigen presentation, depending upon the nature of the particular effector compound and the therapeutic context. Effector compounds include peptides, modified peptides and peptidomimetics. Also disclosed are methods for modulating presentation of an MHC class II restricted antigenic peptide to a T cell. Also disclosed are effector compounds demonstrated to act specifically on a human MHC class II allele. Also disclosed is a second class of compounds, referred to herein as immunomodulatory organic compounds. Such compounds are identified by a method which includes the following steps: providing a first complex comprising an MHC class II molecule to which an antigenic peptide has been bound; contacting the first complex with mammalian Ii key peptide LRMKLPKPPKPVSKMR (SEQ ID NO:1) (or modifications thereof including peptidomimetics), thereby forming a second complex; and screening organic molecules for compounds which bind to the second complex but not to the first complex, and which exhibit immunomodulatory activity. Compounds identified in this manner can be used to modulate an immune response in a mammal.

This is a divisional of application Ser. No. 08/968,676 filed Nov. 12,1997 (now U.S. Pat. No. 5,919,639), which is a divisional of applicationNo. 08/670,605 filed Jun. 26, 1996 (now abandoned).

BACKGROUND OF THE INVENTION

The immune response to specific antigens is regulated by the recognitionof peptide fragments of those antigens by T lymphocytes. Within anantigen presenting cell (APC), peptide fragments of a proteolyticallyprocessed antigen become bound into the antigenic peptide binding siteof major histocompatibility complex (MHC) molecules. These peptide-MHCcomplexes are then transported to the cell surface for recognition (ofboth the foreign peptide and the adjacent surface of the presenting MHCmolecule) by T cell receptors on helper or cytotoxic T lymphocytes. Thatantigen-specific recognition event initiates the immune response cascadefor either protective or deleterious immune responses.

Two classes of MHC molecules function as immune system presenters ofantigenic peptides to T cells. MHC class I molecules receive peptidesfrom endogenously synthesized proteins, such as an infectious virus, inthe endoplasmic reticulum about the time of synthesis of the MHC class Imolecules. The MHC class I-bound antigenic peptides are presented at thecell surface to CD8-positive cytotoxic T lymphocytes, which then becomeactivated and can kill the virus-expressing cells directly. In contrast,MHC class II molecules are synthesized in the endoplasmic reticulum withtheir antigenic peptide binding sites blocked by the invariant chainprotein (Ii). These MHC class II-Ii protein complexes are transportedfrom the endoplasmic reticulum to a post-Golgi compartment where Ii isreleased by proteolysis and a specific antigenic peptide becomes boundto the MHC class II molecule.

The Ii protein is cleaved by intracellular proteases through a series offragments, some of which remain associated with the MHC class IImolecules. This series of fragments has been better defined through thetreatment of cultured, [³⁵S]methionine-labeled cells with certainprotease inhibitors. For example, leupeptin and antipain block theaction of respective classes of proteases on Ii, and on Ii fragmentswhich remain associated with the MHC class II alpha and beta chains. TheMHC class II-bound fragments of Ii are recognized afterimmunoprecipitations with anti-MHC class II antibodies and/or anti-Iiantibodies, gel electrophoresis and autoradiography. In vitro cleavagesof immunopurified MHC class II alpha, beta-Ii protein complexes withcathepsin B, cathepsin D, and other proteases, define site specificcleavages by individual enzymes. The MHC class II alpha, beta chains arerelatively resistant to proteolysis.

These specific cleavage sites in Ii have been confirmed at a molecularlevel with Ii mutants having amino acid replacements at putative sitesfor proteolysis. Several cleavage sites were defined. The crucial sitefor understanding the mechanism of the compounds of this invention is ina region of clustered cationic-hydrophobic dipeptidyl units in human Ii(77-92) (Lu et al, J. Biol. Chem. 145: 899-904, (1990)). Mutation ateach of these four, redundant cleavage sites in the mutant Ii [R⁷⁸→A;K⁸⁰→A; K⁸³→A; K⁸⁶→T] blocks cleavage in that region (Xu et al.,Molecular Immunology 31: 723-731 (1994)).

The region with these clustered, apparent cleavage sites lies in theprimary sequence of Ii about the positions of N-termini of a series ofnaturally occurring Ii fragments, the CLIP peptides. The CLIP peptidesoccur naturally in isolated MHC class II molecules and are abundantlypresented in MHC class II molecules of a mutant cell line which isdeficient in some mechanism which regulates antigenic peptide charginginto MHC class II molecules. This last finding has led to the hypothesesthat the CLIP peptides are an intermediate in peptide charging into MHCclass II molecules (Roche, P., and Cresswell, P., Nature 345: 615-619(1990)), or represent a default pathway to block such molecules fromaccepting ambient peptides after charging with an APC-selected peptidehas failed (Xu et al. in Antigen Processing and Presentation, Humphreys,R. E., ed.: 228-242, Academic Press, NY (1994)).

Overlap among the MHC class II molecule binding sites for antigenicpeptide, the Ii-CLIP peptides, and the therapeutic Ii-key peptide, isbeing determined by x-ray crystallography at a molecular level. Theexact position of influenza virus hemagglutinin peptide HA307-319 in theantigenic peptide binding groove HLA-DR1 was determined first (Stern etal., Nature 368: 215-221 (1994)). Subsequently, the exact positioning ofa CLIP peptide in the same antigenic peptide binding groove wasdetermined (Ghosh et al., Nature 378: 457-462 (1995)). In both cases,the peptides assumed the conformation of a polyprolyl type II helix inthe antigenic peptide binding groove. The backbone atoms of the CLIPpeptide overlay exactly the positions of the backbone atoms of the HApeptide, with comparable placement of side chains into pockets of theMHC class II molecule. Residue position M⁹¹ of the CLIP peptide overlaysthe first residue position of the HA peptide. The CLIP residuesN-terminal to M⁹¹, extending back to P⁸⁷ were also in a polyprolyl typeII helix conformation. More N-terminal residues, including positionshuman Ii L⁷⁷-K⁸³ were not resolved in those crystallographic studies,but clearly lie outside the antigenic peptide binding groove, along theside of the MHC class II molecule.

Thus, although much has been learned with respect to the interaction ofmolecules in the antigen presentation process, the application ofrelevant findings to therapeutic ends remains, for the most part,unrealized.

SUMMARY OF THE INVENTION

The present invention relates, in one aspect, to a class of compoundsreferred to herein as effector compounds. Effector compounds are usefulin connection with the modulation of an immune response. Modulationrefers to the ability of the effector compounds of the present inventionto either enhance (antigen supercharging), or inhibit (immunosuppressantactivities) antigen presentation, depending upon the nature of theparticular effector compound, and the therapeutic context.

Effector compounds include peptides and modified peptides. In apreferred embodiment, the invention relates to the mammalian Ii keypeptide LRMKLPKPPKPVSKMR (SEQ ID NO:1) and modifications thereof, thepeptide YRMKLPKPPKPVSKMR (SEQ ID NO:2) being specifically excluded.Modifications specifically demonstrated include, for example, thedeletion of amino acids from the N-terminus; the deletion of amino acidsfrom the C-terminus; the protection of the C-terminus; the protection ofthe N-terminus; N-terminal extensions; substitutions; and cyclizedderivatives. The invention also encompasses peptidomimetic structureswhich are structurally and functionally related to the effectorcompounds listed above.

Thus, the present invention relates to methods for enhancingpresentation of an MHC class II restricted antigenic peptide to a Tcell. Such methods include contacting the following components underphysiological conditions: an MHC class II expressing antigen presentingcell; the mammalian Ii key peptide LRMKLPKPPKPVSKMR (SEQ ID NO:1) ormodifications thereof (the peptide YRMKLPKPPKPVSKMR (SEQ ID NO:2) beingspecifically excluded); the MHC class II restricted antigenic peptidewhich, when added to the incubation mixture, is not in association withan antigen presenting cell; and a T cell which is responsive to the MHCclass II restricted antigenic peptide.

In another aspect, the present invention relates to methods forinhibiting presentation of an MHC class II restricted antigenic peptideto a T cell. Such methods include contacting the following componentsunder physiological conditions and incubating for an appropriate period:an MHC class II expressing antigen presenting cell displaying on itssurface a T cell-presented epitope from a native protein antigen; andmammalian Ii key peptide LRMKLPKPPKPVSKMR (SEQ ID NO:1) andmodifications thereof (the peptide YRMKLPKPPKPVSKMR (SEQ ID NO:2) beingspecifically excluded).

In other embodiments, the invention relates to effector compounds (i.e.,peptides, modified peptides or peptidomimetics) which induces release ofan antigenic peptide specifically from a human MHC class II allele inthe absence of another antigenic peptide which binds to the human MHCclass II allele. A preferred embodiment is the peptide YRMKLPKSAKPVSQMR(SEQ ID NO:3), or deletion modifications wherein from 0 to 4 amino acidresidues are deleted from the C-terminus and from 0 to 6 amino acidresidues are deleted from the N-terminus.

The invention also relates to an effector compound which induces releaseof a first antigenic peptide from a human MHC class II allele in thepresence of a second antigenic peptide which binds to the human MHCclass II allele. Other specific embodiments include effector compoundswhich bind allosterically to modulate antigenic peptide binding into theantigenic peptide binding site of human MHC class II molecules;allele-specific modulators of antigen presentation; and locus-specificmodulators of antigen presentation.

The present invention also relates to a second class of compounds,referred to herein as immunomodulatory organic compounds. Such compoundsare identified by a method which includes the following steps: providinga first complex comprising an MHC class II molecule to which anantigenic peptide has been bound; contacting the first complex withmammalian Ii key peptide LRMKLPKPPKPVSKMR (SEQ ID NO:1) (ormodifications thereof including peptidomimetics), thereby forming asecond complex; and screening organic molecules for compounds which bindto the second complex but not to the first complex, and which exhibitimmunomodulatory activity. Compounds identified in this manner can beused to modulate an immune response in a mammal.

DETAILED DESCRIPTION OF THE INVENTION

U.S. application Ser. No. 08/064,400, the disclosure of which isincorporated herein by reference, disclosed the fact that a modifiedmammalian Ii key peptide (YRMKLPKPPKPVSKMR) (SEQ ID NO:2) had theability to enhance presentation of an MHC class II restricted antigenicpeptide to a T cell. The present invention is based, in one aspect, onthe surprising discovery that the mammalian Ii key peptide is remarkablytolerant to a broad range of amino acid substitutions, deletions andinsertions. This tolerance was observed in multiple assay contexts,described below, which are intended to mimic a variety of in vivosituations. In addition to this wide range of tolerance, individualpeptides within the group described below were demonstrated to haveremarkable MHC class II species, locus and allele specificities. Giventhe present disclosure, routine experimentation will lead to thedevelopment of novel therapeutic methods which are described more fullybelow. Although the bulk of data reported herein were generated inexperiments employing murine indicator assays for biological activity,the fundamental principles have been extended to studies with purifiedhuman MHC class II molecules (Example 7) and routine experimentationwill permit rapid identification of optimal structures for applicationto the diagnosis and treatment of human diseases.

The wild-type human Ii key peptide is LRMKLPKPPKPVSKMR (SEQ ID NO: 1).In U.S. application Ser. No. 08/064,400, tyrosine (Y) had beensubstituted for the wild-type N-terminal reside, leucine (L). Thesubject invention relates to the mammalian Ii key peptideLRMKLPKPPKPVSKMR (SEQ ID NO:1), as well as modifications thereof, theprior art peptide YRMKLPKPPKPVSKMR (SEQ ID NO:2) being specificallyexcluded. The use of the language “modifications thereof” to describepeptides of the present invention, while indefinite in some contexts, isappropriate given the experimental data described herein whichdemonstrates the many types of modifications which can be made to the Iikey peptide without eliminating its desirable properties.

The experiments described below demonstrated, for example, that thefollowing classes of modifications failed to eliminate certain desirableproperties of the YRMKLPKSAKPVSQMR (SEQ ID NO:3) peptide (a modificationof the peptide LRMKLPKPPKPVSKMR (SEQ ID NO:1)): deletion of amino acidsfrom the N-terminus; deletion of amino acids from the C-terminus;protection of the C-terminus; protection of the N-terminus; N-terminalextensions; substitutions; and cyclized derivatives. In the paragraphswhich follow, the classes of modification will be considered in greaterdetail, as will the assay formats on which the conclusions are based.The desirable properties mentioned above include immunosuppressant (seeExample 6, Tables 29-34) and antigen supercharging activities (seeExamples 1-4, Tables 1-18), depending upon the experimental context.

In addition to peptides and modifications thereof, the present inventionalso encompasses a class of organic compounds commonly referred to aspeptidomimetic structures. Such structures, which demonstrate MHC classII contact points similar to those of the peptides and modified peptidesof the invention, can be identified through routine experimentation.Such compounds may exhibit either equivalent, or superior properties(relative to disclosed peptides). Such properties include, for example,potency, bioavailability and other pharmacokinetic properties, MHC classII locus and allele specificity. Such organic compounds can besynthesized by directed design methods given the structure-functionrelationships revealed in this disclosure and/or through additionalroutine experimental efforts. Such compounds can also be identifiedthrough screening procedures on organic compounds from either existinglibraries of such structures or libraries which are created, forexample, by methods of combinatorial chemistry or genetics. Certainatoms or functional groups in such compounds will overlay, in threedimensional space, atoms or functional groups of active peptides of thetype disclosed herein. Both configurations are structured at the activeregulatory site of contact of such compounds on the MHC class IImolecule, either in a resting or transition state. The class ofcompositions which include both peptides and modified peptides, as wellas structurally-related peptidomimetics, are referred to herein as“effector compounds”.

Examples 1-4 (Tables 1-18) will be briefly considered in order toestablish the concept of tolerance discussed above. These examplesdescribe experimental results observed in the so-called “simultaneous”assay. In the simultaneous assay four principle components are culturedtogether for a 24 hour period. The components of this culture, which areadded simultaneously, are: (a) an antigenic peptide characterized by theability to bind specifically into the antigenic peptide binding grooveof an MHC class II molecule, (b) mitomycin C-treated, MHC classII-positive antigen presenting cells (APC) bearing the MHC class IIallele required for binding of the specific antigenic peptide, andpresentation of the specific antigenic peptide to the antigenicpeptide-specific T cell hybridoma, (c) an effector compound of thepresent invention, and (d) an MHC class II allele-restricted T cellhybridoma specific for the antigenic peptide and the MHC class II allelerestricting its presentation. Following incubation of this primaryculture, an aliquot of its supernatant is transferred into a secondculture well for incubation with an interleukin-dependent lymphoblastoidcell line. The degree of stimulation of that second, indicator cell bythe interleukins which had been released from the activated T cellhybridoma in the primary culture is measured by quantitating tritiatedthymidine deoxyribose {[³H]TdR} uptake into the DNA of the HT-2indicator cells of that second culture.

This situation mimics the in vivo setting in which an antigen presentingcell is contacted with an effector compound of the present invention inthe presence of a second antigenic peptide free in solution. In this invivo context, the effector compound of the present invention stimulatesexchange of the second antigenic peptide for an antigenic peptide boundin the antigen binding groove of MHC class II molecules. Thus, a claimreciting “contacting the above-identified components under physiologicalconditions” is intended to encompass an application in which an effectorcompound of the present invention is administered therapeutically to anindividual.

The results reported in Examples 1-5 demonstrate that the effectorcompounds are characterized by the ability to increase interleukinrelease in the simultaneous assay above the baseline value seen in theresponse to the antigenic peptide without addition of an effectorcompound of the present invention. For example, in Table 1, truncatedhomologs of the peptide YRMKLPKSAKPVSQMR (SEQ ID NO:3) were synthesized,and their biological activity was assayed in in vitro antigenic peptidepresentation assays specific for quantitating T cell hybridomarecognition of antigenic peptides presented by the murine E^(d) andE^(k) MHC class II alleles. Nearly every N- and C-terminal truncation ofthe peptide stimulated interleukin release values exceeding the nopeptide control values (i.e., values determined in the absence of aneffector compound of the present invention in the incubation mixture) inthe murine E^(d) allele experiments. Similar results were observedthroughout Examples 1-4 (Tables 1-18). With an exceptional value fallingbelow the no peptide control value, modifications of the peptideYRMKLPKSAKPVSQMR (SEQ ID NO:3) maintained the ability to stimulateinterleukin release (at least in a locus-specific manner) in thesimultaneous assay which mimics in vivo therapeutic administration of anantigenic peptide together with an effector compound of the presentinvention.

The specific peptide modifications reported in Table 1 includedN-terminal deletions of up to about 7 amino acid residues; C-terminaldeletions of up to about 6 amino acid residues; as well as N- andC-terminally protected variations of the N- and C-terminal deletions.Table 2 reports data from N-terminal extension experiments. In thistable, data from extensions of up to 6 amino acid residues werereported. Tables 3-10 report data from substitution studies whereinL-amino acid residues in peptides were substituted with other L-isomeramino acids or modified L-isomer amino acids. Table 11-12 report datafrom studies in which D-isomer amino acids were substituted for selectedL-isomer amino acids. Table 13 reports N-methyl amino acid substitutiondata. Table 14 reports N-methyl substitution data, with some peptidesincluding D-isomer amino acid substitution together with N-methylsubstitution in a single peptide. Tables 15-18 relate to multiplesubstitutions, position 5 substitutions and cyclical analogs.

The remarkable observation made in connection with the manymodifications reported in Examples 1-4 (Tables 1-18) is that in very fewinstances was the stimulated interleukin release observed for thesubstituted peptides, less than the no peptide control. The substitutionof aspartate (D) or glutamate (E) for an amino acid found in a wild-typemammalian Ii sequence represents an exception to this observation whichwas observed in several experiments. The observed locus and allelespecificity is discussed more fully below.

Although, as discussed above, interleukin release in the simultaneousassay is generally elevated above no peptide control with nearly allsubstituted peptides, certain peptides can be identified throughanalysis of the data which perform substantially better than others. Oneof skill in the art would predict with a high degree of certainty thatsimilar screening assays conducted using human, rather than murine MHCclass II alleles, would identify effector compounds exhibiting effectssimilar to those observed in connection with the murine alleles. Theidentification of such effector compounds is a matter of routineexperimentation, given the present disclosure.

The effector compounds of the present invention find application in avariety of in vitro and in vivo therapeutic contexts. Generally, themethods are applied either for the purposes of immunosuppression orantigen supercharging.

Antigen supercharging is accomplished using the effector compounds ofthe present invention by exploiting both the “antigen spilling” and the“antigenic peptide binding” properties of the compositions discussedabove. Antigen spilling refers to the ability of the effector compoundsto remove antigenic peptide from the antigenic peptide binding groove ofMHC class II molecules on the surface of antigen presenting cells. Theantigenic peptide binding property refers to the facilitation (byeffector compounds) of the binding of a second peptide withimmunomodulatory properties into the antigen peptide binding groove ofMHC class II molecules. Thus, effector compounds having the ability tostimulate the ejection of antigenic peptides from MHC class II, arecontacted with antigen presenting cells in the presence of a secondantigenic peptide. The object of the therapeutic approach is tostimulate the exchange of the second antigenic peptide for the antigenicpeptide which is prebound, in vivo, to the antigen peptide bindinggroove of MHC class II.

As indicated above, the methods of the present invention include both invitro and in vivo embodiments. In vitro, antigen presenting cellsisolated from an individual (e.g., lymphocytes) are treated byincubating the cells in a solution containing appropriate concentrationsof an effector compound (characterized by the ability to spill antigenicpeptide either in the presence of a second antigenic peptide or in theabsence of a second antigenic peptide) together with appropriateconcentrations of a second peptide. Again, the goal of the in vitroincubation is to substitute the second peptide for the first peptide inthe peptide binding groove of MHC class II molecules on the surface oflymphocytes following stimulation of the ejection of the first peptide.Following treatment of the cells in vitro, they are reinfused into theindividual at which time T cells responsive to the second antigenicpeptide will be presented with the antigen and an immune response willbe stimulated against the second antigenic determinant.

For example, antigen presenting cells originating from a patient can becontacted in vitro with a solution containing a tumor vaccine peptidetogether with an effector compound of the present invention which aidsin the exchange of the tumor vaccine peptide for an antigenic peptidefound in association with MHC class II on the surface of the cellsoriginating from the patient. In the case of malignant antigenpresenting cells (such as some cells of the lymphoma or melanoma classesof malignancies) such antigenic peptide-primed cells can be renderedincapable of proliferating prior to reinfusion into the patient.

Another application for the effector compounds of the present inventionfor the enhancement of immunity against cancer determinants, is the invitro treatment of malignant cells taken from a patient, with aneffector compound. The effector compound of the invention, through anaction on intracellular processing and binding of endogenoustumor-associated determinants in a malignant cell, enhances binding tothe intracellular MHC class II molecules of endogenous tumor-associateddeterminants prior to their surface expression on the cell. That is, bydisrupting the occupancy by Ii or Ii fragments of the antigenic peptidebinding site of MHC class II molecules, additional endogenous peptidescome to be bound in MHC class II molecules and subsequently presented.In that manner the patient will become primed to both more and a widerrange of endogenous tumor-associated determinants than available throughcurrent cancer vaccine peptide immunization schemes.

Another use of the effector compounds of the present invention is toenhance in vitro peptide charging of antigen presenting cells for thepurpose of developing either cloned T cell lines or of T cellhybridomas, all being of therapeutic or diagnostic value. To this end,the more efficient identification of autoimmune disease-related,antigenic peptides is made possible. In studies by others, antigenicpeptides were acid-eluted from immunopurified MHC class II moleculesfrom antigen presenting cells obtained from clinical material. AfterHPLC separations, some of those peptides were tested for biologicalactivity in vitro. The activities of trace quantities of such peptidescan be enhanced greatly by the adjuvant effect of the effector compoundsof the present invention.

Related to that use to characterize naturally occurring, disease-relatedpeptides from immunopurified MHC class II molecules is the use ofeffector compounds to release antigenic peptides, or a subset ofantigenic peptides, from such MHC class II molecules.

In vivo, the antigen spilling effector compound is coadministered withthe second antigenic peptide at concentrations appropriate for thesupercharging of MHC class II molecules on the surface of antigenpresenting cells with a second antigenic peptide. The effector compoundand the second antigenic peptide can be administered throughconventional delivery modes including, for example: intramuscularinjection, oral, intranasal or buccal administration, through the use ofa subcutaneous implant wherein the release is controlled, or throughdirect injection into a locally inflamed space (e.g., a joint).

As mentioned above, the present invention relates primarily to twotherapeutic modalities-antigen supercharging and immunosuppressantactivities. With respect to antigen supercharging, compositions of thepresent invention can be applied to immunotherapy of allergic disease.For example, immunization with an allergy suppressing peptide, such asthose which have been described for the treatment of allergy to ragweedor cat dander, can be enhanced by coinjection in a formulationcontaining both the allergy suppressing peptide and an effector compoundof this invention selected to be effective in augmenting presentation ofantigenic peptides by human MHC class II molecules. The effectorcompound would enhance presentation of the allergy suppressing antigenby a mechanism similar to that which takes place in the simultaneousassays, as reported in the Examples.

Similarly, the effector compounds can be applied in the therapy ofmalignant disease. For example, immunization with amalignancy-associated peptide, such as a malignant melanoma specificpeptide, can be enhanced by coinjection with an effector compound toaugment presentation of antigenic peptides by human MHC class IImolecules.

In addition to applications related to antigen supercharging, thepresent invention relates also to immunosuppressant methods. Asdiscussed above in connection with the antigen superchargingembodiments, the immunosuppressant embodiments may be practiced in vitroor in vivo. The formulation and delivery methods are substantiallysimilar for both the immunosuppressant and antigen superchargingembodiments, but for the fact that no second antigenic peptide isincluded in immunosuppressant embodiments. Rather, the antigen spillingproperty of the effector compounds are exploited to remove anautodeterminant peptide from the MHC class II antigen peptide bindinggroove. Immunosuppression is the clinical effect, although the antigenicpeptide binding groove might be filled by an ambient peptide.

Effector compounds of the present invention, when found to be effectivein certain human MHC class II alleles (with or without relative allelespecificity of action) can be applied to the treatment of disease. Forexample, in the case of autoimmune disease, effector compounds can beadministered to affected individuals in a manner appropriate for thesuppression of presentation of disease-related antigenic determinants.Such administration might be systemic by oral, intravenous,intramuscular, subcutaneous, intraperitoneal routes, or by localinjection, such as into an inflamed joint. Systemic and/or local controlof disease can thus be achieved. Such therapeutic control is achieved bycontacting the patients' inflammatory or other antigen presenting cellswith an effector compound in a manner sufficient to effect thenonstimulating state as revealed in vitro in assays of the Examples.Specific examples of human autoimmune disease suited for therapyinclude, for example, rheumatoid arthritis, multiple sclerosis, diabetesmellitus, lupus erythematosus and psoriasis.

Certain effector compounds can be applied to suppress allograftrejection. Systemic treatment of antigen presenting cells of anallograft recipient with such compositions, as subsequently chosen toregulate presentation of antigenic peptides by a wide range of human MHCclass II molecules, would lead to immunosuppression. Preferredcompositions include the cyclical AE381, a cyclical form of the sequenceLRMKLPK (SEQ ID NO:4), joined through an amido bond from the N-terminalamino group to the C-terminal carboxyl group of the peptide, andhomologs which suppress the antigenic peptide prepulse assay withouteffecting antigen supercharging in the simultaneous assay.

Selected effector compounds of the present invention were determined toexhibit MHC-class II allele-specificity. MHC class II allele-specificityrefers to the preferential interaction of compounds of given chemicalstructures with one MHC class II allele as compared to interaction witha second MHC class II allele at the same genetic locus. The expression“allele-specificity”, as used herein, refers to a differential in thesimultaneous assay described below, of at least about 2-fold. Forexample, in Tables 3-10 of Example 2, shown below, particular amino acidsubstitutions were identified which exhibited a high degree of activityon the E^(d) allele as compared with the E^(k) allele. In addition,other modifications were determined to exhibit a high degree of activityon the E^(k) allele as compared with the E^(d) allele. Thus, for anygiven Ii key peptide, one of skill in the art would predict with a highdegree of certainty that an allele-specific homolog could be generatedthrough the use of routine experimentation (e.g., amino acidsubstitution analysis). While these principles have been established instudies with murine MHC class II alleles, it is predictable with a highdegree of certainty that such fundamental principles will extend tostudies carried out using human alleles.

A high degree of locus specificity was also observed in the studiesreported below. For example, in the legend to Table 3, it is stated thatno activity was observed in experiments involving the A locus.Furthermore, while not specifically reported herein, in the experimentssummarized in Tables 3-10, parallel experiments were conducted with theA locus alleles. In every instance, the compositions of the presentinvention exhibited less than 15% activity when carried out with A locusalleles, as compared with otherwise identical studies carried out with Elocus alleles.

Allele- and locus-specific effector compounds are useful, for example,in connection with in vivo and in vitro therapeutic applications of thetype described above. The fact that effector compounds are significantlymore active on one, or a few, MHC class II alleles at one genetic locus,as compared with other alleles at that same genetic locus, hastherapeutic implications. This is also true with respect to observationsof significantly more activity in connection with alleles at one geneticlocus, as compared to alleles at a second genetic locus. Suchdifferential activity on one or a few alleles at one genetic locusand/or one genetic locus of MHC class II molecules, leads to a morefavorable therapeutic index. The therapeutic index is the ratio of theeffective therapeutic dose to the dose at which a significant toxicityis observed. Effector compounds of this invention, being active at onlya subset of the MHC class II molecules which are responsible forpresentation of antigenic epitopes to the T lymphocytes which regulatethe disease process, can regulate that disease process selectively whileMHC class II products of other, relatively unaffected alleles or lociremain available to present epitopes from common infectious agents. Manyautoimmune diseases which demonstrate familial patterns of inheritance,have been shown to be linked genetically to alleles of the MHC Class IImolecules. For example, forms of rheumatoid arthritis are linked tocertain alleles of the HLA-DR4 allele of MHC class II molecules.Blocking inflammatory responses through that allele specifically, can bepredicted to suppress the inflammatory response while leaving availableMHC class II molecules from other alleles and genetic loci forprotection against common infections. A significant side effect ofcurrent cytotoxic, immunosuppressive therapies for rheumatoid arthritisis generalized immunosuppression (regardless of MHC class II allelespossessed by the patient). Achieving MHC class II allele and/or locusspecificity is therefore of considerable clinical value in treatingpatients with autoimmune disease. Parallel arguments for the value ofthe specificity of action of effector compounds of this invention can bemade for their use in controlling MHC class II allele-related responsesto allergens and vaccine peptides.

As disclosed herein, the effector compounds of the present invention areeffective in the modulation of the immune response for the purposeseither of inducing immunosuppression or of enhancing theimmunomodulatory capacity of a second antigenic peptide which isintroduced into the MHC class II molecules by the antigen superchargingproperty of the effector compounds of the invention. The effectorcompounds of this invention can also be applied to the discovery of anadditional class of organic compounds which act by becoming bound intothe antigenic peptide binding groove. Such additional compounds arecharacterized by the ability to bind into a MHC class II molecule withsuch a tight affinity, as to inactivate the biological function of theMHC class II molecule with potency of a covalent inhibitor. Suchcompounds are referred to herein as “immunomodulatory organiccompounds”, to avoid confusion with the “effector compounds” of theinvention (which also possess immunomodulatory characteristics).

Screening such immunomodulatory organic compounds using differing MHCclass II alleles can identify MHC class II allele specificity.Immunomodulatory organic compounds which exhibit this type of allelespecificity can be used to inactivate the biological activity of subsetsof MHC class II molecules associated with particular autoimmune or otherdiseases. Parenthetically, it is noted that although significant degreesof MHC class II allele specificity have been disclosed in theexperiments described herein, yet greater degrees of MHC class II alleleactivity for therapeutic purposes will likely be achieved through theuse of the effector compounds of the invention to potentiate the binding(and identification) of this second class of compounds, theimmunomodulatory organic compounds.

The immunomodulatory organic compounds are identified by screeningcollections of organic compounds for their specific binding to MHC classII molecules to which an effector compound of the present invention hasbeen specifically bound, relative to binding to control MHC class IImolecules to which no effector compound has been bound. The experimentsdisclosed herein demonstrate the stable association of certain effectorcompounds with MHC class II molecules. Such stable complexes demonstratealtered conformations through the increased lability of bound antigenicpeptides and the increased facility of binding of second antigenicpeptides. Immunomodulatory organic compounds identified by the methodsdescribed herein can be used in therapeutic contexts to alter an immuneresponses. Such compounds would be administered to individuals bysystemic or parenteral routes and would contact MHC molecules withinthose individuals. Alternately, such compounds would be administered informulations with effector compounds of this invention in a fashion inwhich the effector compound would, upon contacting a MHC class IImolecule on an antigen presenting cell, facilitate the binding of theimmunomodulatory organic compound.

EXAMPLES Example 1 Identification of the Shortest, Most Active AE101Series Sequence

Three in vitro assays of the effects of AE101 series compounds (alsoreferred to as effector compounds) on presentation of antigenic peptidesare used in the experiments presented in these Examples. These threeassays test in various ways the molecular mechanism of action of thesubject compounds. The assays are the “simultaneous assay”, the “peptideprepulse assay”, and the “processed antigen assay”.

In the “simultaneous assay” the four components of the assay arecultured together for a 24 h period. The components of this primaryculture, added at the same time or simultaneously, are: (a) theantigenic peptide, (b) mitomycin C-treated, MHC class II-positiveantigen presenting cells (APC) with the MHC class II allele required forbinding of the specific antigenic peptide and its presentation to theantigenic peptide-specific T cell hybridoma, (c) an AE101 serieseffector peptide, and (d) MHC class II allele-restricted T cellhybridoma specific for the antigenic peptide and the MHC class II allelerestricting its presentation. At the end of the incubation of thisprimary culture, an aliquot of its supernatant is transferred into asecond culture well for incubation with an interleukin-dependentlymphoblastoid cell line. The degree of stimulation of that second,indicator cell by the interleukins which had been released from theactivated T cell hybridoma in the primary culture is measured byquantitating tritiated thymidine deoxyribose {[³H]TdR} uptake into theDNA of the HT-2 indicator cells of that second culture.

In a second type of assay, the “peptide prepulse assay”, the antigenicpeptide is incubated with paraformaldehyde-fixed APC for 6 h. The APCare washed and incubated for 24 h with the AE101 series homolog and theT cell hybridoma specific for the antigenic peptide. After thatincubation, an aliquot of the culture supernatant is transferred to asecond culture to measure the relative degree of T hybridomastimulation, as reflected in the effect of released interleukins on thegrowth of an interleukin-dependent, indicator cell line, as describedabove.

The two above described assays measure different aspects of themolecular mechanism of the AE101 series of peptides. In the“simultaneous assay”, AE101 series peptides are thought to induce therelease of endogenously bound peptides and to permit the binding of thesecond, specific antigenic peptide which is relatively abundant in theculture fluid. The AE101 series peptides enhance antigenicpeptide-specific T cell responses in simultaneous assays. In the“peptide prepulse” assay, the specific antigenic peptide becomes boundto MHC class II molecules on the surface of the fixed APC during the 6 hprepulse incubation. The effect of the AE101 series homologs is thenthought to release that antigenic peptide, resulting in an apparentsuppression of the immune response.

The AE101 series peptides are thought to contact the MHC class IImolecules at a discrete site outside the antigenic peptide-bindinggroove. AE101 peptide binding to this “Ii-KEY” site is thought to inducea conformational change in the MHC class II molecules accelerating thedissociation of previously bound antigenic peptide. The released,specific antigenic peptide is of such low concentration after releasethat its rebinding is effectively prevented by dilution in thesurrounding culture medium. The AE101 series peptides thus inhibitantigenic peptide-specific T cell responses in the “peptide prepulse”assays.

In a third type of assay, the “processed antigen assay”, certain of theAE101 series of peptides inhibit stimulation of specific T cellhybridomas by antigenic peptides which are derived from the endogenousprocessing of a native protein antigen. This assay, which is related tothe peptide prepulse assay, is performed by incubating APC with nativeprotein antigen for 8 h, after which the pulsed APC are washed andtreated with mitomycin C. Those pulsed APC are then combined with AE101series peptides and T cell hybridomas and are incubated for 24 h. Afterthat incubation, an aliquot of the culture supernatant is transferred toa second culture to measure T cell stimulation, as reflected in theeffect of released interleukins on the growth of aninterleukin-dependent, indicator cell line. During the 8 h incubationwith native protein antigen, the protein antigen is taken into the APC.The native protein enters the processing pathway within the APC, whereit is enzymatically cleaved to peptide fragments. Those peptidefragments with high affinity for the particular MHC class II moleculesproduced by the APC form antigenic peptide/MHC class II complexes whichare transported to the cell surface. At the cell surface, the MHC classII molecules are contacted by the AE101 peptides, which cause therelease of the antigenic peptide by the same mechanism proposed for the“peptide prepulse” assay above.

For these various assays, the following antigenic peptides were used:HEL11-25, hen egg lysozyme 11-25, AMKRHGLDNYRGYSL(A^(d)) (SEQ ID NO:5);HEL46-61, hen egg lysozyme 46-61, NTDGSTDYGILQINSR(Ak) (SEQ ID NO:6);HEL106-116, hen egg lysozyme 106-116, NAWVAWRNRCK (E^(d)) (SEQ ID NO:7);PGCC81-104, pigeon cytochrome c 81-104, IFAGIKKKAERADLIAYLKQATAK (Ek)(SEQ ID NO:8), and THMCC82-103, tobacco hornworm moth cytochrome c82-103, FAGLKKANERADLIAYLKQATK (Ek) (SEQ ID NO:9). AE101 series peptideswere obtained from commercial sources. In general, the purity andcomposition of each peptide was confirmed by HPLC separation and massspectrometry. The native protein antigens were HEL, hen egg lysozyme,and PGCC, pigeon cytochrome C. They were obtained from commercialsources.

In all assays antigenic peptides and the AE-101 series peptides weredissolved in phosphate-buffered saline (PBS; 0.01 M sodium phosphatebuffer, pH 7.2, 0.1 M NaCl). The solutions were sterilized byfiltration, and the peptide concentrations were determined by amino acidanalysis (Applier Biosystems, Inc. 420A/130A derivatizer/HPLC afterhydrolysis with 6 N HCl for 24 h in vacuo).

For the experiments of these Examples, several T cell hybridomas, whichare specific for certain antigenic peptides, were used. The TPc9.1 Thybridoma is specific for pigeon cytochrome C 81-104 peptide presentedon the murine class II MHC allele E^(k). The TPc9.1 hybridoma respondsheteroclitically to tobacco hornworm moth cytochrome c 82-103 on E^(k).The 3A9 T hybridoma is specific for hen egg lysozyme 46-61 on A^(k). The9.30.B2 hybridoma is specific for hen egg lysozyme 11-25 on A^(d), andthe G28.C9 hybridoma is specific for hen egg lysozyme 106-116 on E^(d).The A20 and CH27 B cell lymphoma lines express H-2^(d) and H-2^(k)alleles, respectively.

Antigenic peptide-specific T cell activation was measured by thefollowing procedure. Mitomycin C-treated A20 cells (A^(d)E^(d)) or CH27cells APC (A^(k)E^(k)) were generated by incubating 5×10⁶ cells/mL for20 min at 37° C. with 0.025 mg/mL of mitomycin C (Sigma) in Dulbecco'sModified Eaglel's Medium (DMEM)/10 mMN-2(hydroxyethylpiperazine-N″[2-ethanesulfonic acid] (HEPES), followedby two washes with four volumes of DMEM-5% fetal calf serum (FCS), 10 mMHEPES. Fixed APC were generated by treating 1×10⁶ cells/mL for 5 minwith 0.5% paraformaldehyde in PBS (pH 7.2), followed by two washes withfour volumes of DMEM-10% FCS, 10 mM HEPES. T cell hybridomas wereirradiated 2200 rads before each assay.

For the “simultaneous assay”, 5×10⁴ mitomycin C-treated APC, 5×10⁴ Thybridoma cells and a submaximal concentration of antigenic peptide werecultured with and without serial 4-fold dilutions of each AE101 seriespeptide, usually at 64 μM, 16 μM, 4 μM, and 1 μM, at pH 7.2-7.4, incomplete DMEM-5% FCS, 10 mM HEPES, 1×nonessential amino acids (Sigma), 1mM sodium pyruvate, 2 mM L-glutamine, 100 U/mL penicillin G, 100 μg/mLstreptomycin sulfate, 5×10⁻⁵ M 2-mercaptoethanol (2-ME). Wellscontaining only T hybridoma cells (T)+APC were included to monitor forbackground T cell activation; and wells containing T+APC+AE101 seriespeptide were included to monitor for non-specific T hybridoma activationby each AE101 series peptide. Supernatants (aliquots of 20, 40 or 75 μl)from each culture were removed after 24 h and were assayed for theireffect on growth of 1×10⁴ interleukin-dependent HT-2 lymphoblastoidcells (added in 140, 120 or 75 μl complete Roswell Park MemorialInstitute (RPMI) 1640 buffer—5% FCS, respectively), as measured byincorporation of [³H]TdR, added at 1 μCi/well during the last 5 h of a24 h HT-2 assay. For all assays the reported value is the mean oftriplicate wells, with a mean standard error of less than +10%. Sincethe degree of stimulation varied among assays, usually both in theprimary culture and in the secondary HT-2 indicator culture, forcomparisons among assays performed at different times, standard orreference peptides were always included.

The “peptide prepulse assay” was carried out under essentially the sameconditions as described for the “simultaneous assay” with the followingmodifications. Fixed APC were first incubated for 6 h at 1×10⁶ cells/mLin complete DMEM-5% FCS in 24-well microculture plates (1 mL/well) withantigenic peptide, followed by four washes with 10 volumes of DMEM-5%FCS. The cells were then exposed to varying concentrations of AE101series peptide (64 μM, 16 μM, 4 μM, and 1 μM) for 24 h in the presenceof the T cell hybridoma specific to the antigenic peptide. Interleukinrelease from these cultures was measured by proliferation of HT-2 cellsto interleukins in supernatants transferred from the primary culture.Generally, a single dose of 64 μM of each AE101 series peptide was used.The baseline T cell response was measured by culturing T hybridoma cellswith the antigenic peptide-prepulsed APC in the absence of AE101 seriespeptides.

The “processed antigen assay” was carried out under essentially the sameconditions as the “peptide prepulse assay”, with the followingmodifications. Untreated APC were incubated at 1×10⁶/mL in 24-wellplates (1 mL/well) with native protein antigen for 8 h. Following thatincubation, the pulsed APC were washed, treated with mitomycin C, andwashed again. AE101 series peptide was added at 64 μM, 16 μM, 4 μM, and1 μM concentrations for 24 h in the presence of the T cell hybridomaspecific for the antigenic peptide. Interleukin release from thesecultures was measured by proliferation of HT-2 cells to interleukins insupernatants transferred from the primary culture. The baseline T cellresponse was measured by culturing T hybridoma cells with the nativeantigen-prepulsed APC in the absence of AE101 series peptides.

In order to define the shortest AE101 series peptide with the maximalactivity, a series of N- and C-terminally truncated homologs of AE101was synthesized (Table 1). The biological activities of these peptideswere assayed in in vitro antigenic peptide presentation assays specificfor quantitating T cell hybridoma recognition of certain antigenicpeptides presented by the murine E^(d) and E^(k) MHC class II alleles.The assays used were (1) the “simultaneous assay”, and (2) the “peptideprepulse assay”.

TABLE 1 N- and C-Terminal Truncation Analogs of AE101. Peptide SequenceE^(d) E^(k) None  1.0  1.0 AE101 YRMKLPKSAKPVSQMR 13.6, 4.9  1.0, 0.8(SEQ ID NO:3) AE102 RMKLPKSAKPVSQMR 13.3, 4.2  1.0, 0.8 (SEQ ID NO:10)AE103 KLPKSAKPVSQMR  3.4, 1.3  0.7, 0.7 (SEQ ID NO:11) AE104 PKSAKPVSQMR 2.6, 0.9  0.8, 1.1 (SEQ ID NO:12) AE105 SAKPVSQMR  4.5, 1.1  0.7, 0.8(SEQ ID NO:13) AE106 YRMKLPKSAKPVSQ 16.9, 4.4  2.0, 0.9 (SEQ ID NO:14)AE107 YRMKLPKSAKPV 21.7, 4.8  1.0, 0.8 (SEQ ID NO:15) AE108 YRMKLPKSAK32.0, 11.6  1.2, 0.9 (SEQ ID NO:16) AE109 Ac-YRMKLPKSAK-NH₂ 39.3, 20.8 6.9, 2.2 (SEQ ID NO:16) AE110 Ac-LRMKLPKSAK-NH₂ 47.1, 27.8  7.6, 2.1(SEQ ID NO:17) AE167 Ac-LRMKLPKPPP-NH₂ 20.1, n.d.  3.4, n.d. (SEQ IDNO:18) AE168 Ac-LRMKLPKPPK-NH₂ 16.7, n.d.  4.7, n.d. (SEQ ID NO:19)AE111 Ac-YRMKLPKSA-NH₂ 39.2, 18.8  7.2, 2.0 (SEQ ID NO:20) AE112Ac-YRMKLPKS-NH₂ 42.8, 26.2 15.3, 3.2 (SEQ ID NO:21) AE113 Ac-YRMKLPK-NH₂36.3, 23.1 15.5, 6.7 (SEQ ID NO:22) AE114 Ac-LRMKLPK-NH₂ 39.8, 26.015.9, 3.9 (SEQ ID NO:23) AE115 Ac-YRMKLP-NH₂ 19.9, 5.7 18.6, 3.8 (SEQ IDNO:24) AE116 Ac-YRMKL-NH₂  7.1, 2.1 15.5, 3.2 (SEQ ID NO:25) AE117Ac-YRMK-NH₂  2.3, 1.0 14.6, 10.6 (SEQ ID NO:26) AE118 Ac-YRM-NH₂  1.0,0.6  5.6, 3.0 (SEQ ID NO:27)

Table 1: Activities of N- and C-terminal truncation analogs of AE101peptide in a simultaneous assay. Activities for each allele are given as“Times Baseline Response” for assays with the E^(d) and E^(k) alleles.For this simultaneous assay, MHC class II-positive APC, treated withmitomycin C, were incubated with an antigenic peptide-specific T cellhybridoma, the respective antigenic peptide, and an AE101 seriespeptide. The concentrations of antigenic (Ag) peptides were 0.4 μM ofHEL106-116 for E^(d) and 0.075 μM of THMCC82-103 for E^(k). The AE101series peptides were used at 64 μM (first value) and 16 μM (secondvalue) for E^(d) and E^(k). Interleukin released from the T hybridomacells was quantitated after 24 h by [³H]TdR incorporation in interleukindependent HT-2 cells. The dilutions of primary culture supernatant takeninto the HT-2 cell assay were 1:4 for E^(d) and 1:2 for E^(k). Theobserved response, “Times Baseline Response”, equaled CPM of (T+APC+Agpeptide+AE101 series peptide)/CPM of (T+APC+Ag peptide). The means oftriplicate wells had an average SEM of ≦10%. The T cell response toantigenic peptide alone was designated as the baseline value 1. “Nopeptide” was an assay without AE101 series peptide. The single letteramino acid codes used throughout all Tables are as follows: A=L-alanine,Cit=L-citrulline, D=L-aspartate, E=L-glutamate, F=L-phenylalanine,H=L-histidine, Harg=L-homoarginine, K=L-lysine, k=D-lysine, L=L-leucine,l=D-leucine, mL=n-methyl-L-leucine, M=L-methionine, m=D-methionine,N=L-asparagine, Orn=L-ornithine, P=L-proline, p=D-proline,hydrP=L-hydroxyproline, R=L-arginine, r=D-arginine, Q=L-glutamine, andY=L-tyrosine. Whenever mL appears in a table, it is set off by bracketsto lessen confusion with “D-methionine, L-Leucine”. Likewise, wheneverHarg, Cit, Orn occur in a table, they are set off by spaces to lessenconfusion, for example, with “L-histidine, D-alanine, D-arginine,D-glycine” etc.

These assays revealed the activity of the AE114 peptide which containedthe 7 amino acid primary sequence of murine Ii76-91 and human Ii77-92,respectively (sequences in both species being identical), withN-terminal acetylation and C-terminal amidation. While AE114 is activeon both the murine class II MHC E^(d) and E^(k) alleles, the shortertetrapeptide AE117 retained full activity on E^(k) but not on Ed. Forthe E^(d) allele, the shortest peptide analog retaining the maximalobserved activity was the 7-mer, AE114. Replacing the N-terminaltyrosine (Y) by leucine (L; the wild type residue) slightly increasedthe potency of the peptide in the E^(d) system (AE101>AE109, andAE114>AE113). Blocking the N- and C-termini increased the potency of theAE108 peptide in both the E^(d) and E^(k) systems: AE109>AE108.

TABLE 2 N-Terminal Extension Analogs of AE110. Peptide Sequence E^(d)E^(k) A^(d) A^(k) None 1.0 1.0 1.0 1.0 AE109 Ac-YRMKLPKSAK-NH₂ 25.5 43.70.9 1.2 (SEQ ID NO:16) AE110 Ac-LRMKLPKSAK-NH₂ 23.8 19.1 0.7 0.8 (SEQ IDNO:17) AE155 Ac-SLRMKLPKSAK-NH₂ 23.4 8.4 0.8 1.0 (SEQ ID NO:28) AE154Ac-DSLRMKLPKSAK-NH₂ 15.1 2.2 1.1 1.0 (SEQ ID NO:29) AE153Ac-LDSLRMKLPKSAK-NH₂ 8.5 1.4 1.2 1.0 (SEQ ID NO:30) AE152Ac-QLDSLRMKLPKSAK-NH₂ 2.3 0.4 0.9 0.5 (SEQ ID NO:31) AE151Ac-LQLDSLRMKLPKSAK-NH₂ 2.5 0.6 1.1 0.5 (SEQ ID NO:32) AE150Ac-NLQLDSLRMKLPKSAK-NH₂ 3.2 0.8 1.0 0.0 (SEQ ID NO:33)

Table 2: Activities of N-terminally extended AE101 series peptides in asimultaneous assay. Activities (Time Baseline Response) for enhancementof antigen presentation for each indicated allele were determined in asimultaneous assay carried out as described in the legend of Table 1,with the following modifications. The concentrations of antigenicpeptides were 0.05 μM of HEL46-61 for A^(k) and 0.05 μM of HEL11-25 forA^(d). The concentrations of antigenic peptides were 0.05 μM ofPGCC81-104 for E^(k) and 0.05 μM of HEL106-116 for E^(d). Theconcentration of AE101 series peptides was 64 μM in all four allelicsystems. A 1:2 dilution of supernatant of the primary culture was takenfor the HT-2 cell assays for all four allelic systems. The N-terminalextensions in peptides AE150 through AE155 are wild-type residues frompositions from N⁷⁰ to S⁷⁵ in the amino acid sequence of murine Ii.

In the E^(d) and E^(k) allelic systems, extending the N-terminus of theAE110 peptide with additional wild-type sequence of murine Ii resultedin a systematic decrease in the enhancing activity in the “simultaneous”type of assay. In the E^(k) system in particular, such N-terminalextension finally led to inhibition (AE152 and AE151). In the A^(d)system, while AE110 was not active, such N-terminal extensions also“uncovered” no activity. In the A^(k) system, where the AE110 referencepeptide was inactive, addition of N-terminal wild-type sequence led toinhibitory peptides: AE152, AE151, and AE150.

In summary, the experiments of this Example demonstrate the shortestactive AE101 series peptides, acceptance in an in vitro assay of N- andC-terminal protection against exopeptidases, and significant MHC ClassII allele specificity of certain peptides as a function of peptidelength.

Example 2 L-Isomer Amino Acid Substitutions at 5 Positions in AE114 and2 Positions in AE109 (a Longer Analog of AE114) Indicate Side ChainPreferences for Potency and for Allele-Specificity

Amino acid substitutions at 5 positions in AE114 (a 7-amino acidpeptide) and 2 positions in AE109 (a 10-amino acid peptide) definedpreferences for certain side chain structures at each of thosepositions.

TABLE 3 Substitution Series At Leucine⁷⁶ (Position 1) in AE114. PeptideSequence Ed Ek None 1.0 1.0 AE114 Ac-L RMKLPK-NH₂ 10.0 26.9 (SEQ IDNO:23) AE180 Ac-Orn RMKLPK-NH₂ 12.6 10.8 (SEQ ID NO:34) AE181 Ac-CitRMKLPK-NH₂ 9.0 11.5 (SEQ ID NO:35) AE182 Ac-HArg RMKLPK-NH₂ 18.8 26.5(SEQ ID NO:36) AE183 Ac-H RMKLPK-NH₂ 10.3 17.3 (SEQ ID NO:37) AE184 Ac-KRMKLPK-NH₂ 14.1 11.8 (SEQ ID NO:38) AE185 Ac-D RMKLPK-NH₂ 1.2 0.9 (SEQID NO:39) AE186 Ac-E RMKLPK-NH₂ 2.5 1.3 (SEQ ID NO:40) AE187 Ac-NRMKLPK-NH₂ 6.3 9.5 (SEQ ID NO:41) AE188 Ac-Q RMKLPK-NH₂ 8.8 7.7 (SEQ IDNO:42) AE189 Ac-F RMKLPK-NH₂ 12.1 18.2 (SEQ ID NO:43) AE113 Ac-YRMKLPK-NH₂ 12.3 24.9 (SEQ ID NO:22) AE190 Ac-M RMKLPK-NH₂ 9.8 15.2 (SEQID NO:44)

Table 3. Activities of substitution series at Leucine⁷⁶ in AE114 in asimultaneous assay. The data are from assays (described in Example 1) inwhich the concentrations of AE101 series peptide was 64 μM for eachallele. The supernatant dilution taken into the HT-2 cell assay was 1:4for each allele. To compare results between the two systems more easily,the values for the 64 μM AE101 series peptide in the E^(k) system werereduced relative to the E^(d) system by a factor of 10; since thebaseline CPM for E^(k) was approximately 0.1 times the baseline CPM forE^(d).

In the E^(d) allele, AE114 homologs with HArg, K, Orn, Y, and F at thefirst position generated peptides with high activities. The leastactivity was found in homologs with negatively charged residues D and Eat that position. In the E^(k) system, the five amino acid substitutionsat the first position in AE114 with high activities were L, HARG, Y, F,and H. The two substitutions with least activity in the E^(k) systemwere D and E.

TABLE 4 Substitution Series At Arginine⁷⁷ (Position 2) in AE109. PeptideSequence E^(d) E^(k) None 1.0 1.0 AE109 Ac-Y R MKLPKSAK-NH₂ 3.3 2.3 (SEQID NO:16) AE121 Ac-Y A MKLPKSAK-NH₂ 1.1 1.0 (SEQ ID NO:45) AE130 Ac-YOrn MKLPKSAK-NH₂ 0.9 1.1 (SEQ ID NO:46) AE131 Ac-Y Cit MKLPKSAK-NH₂ 2.80.8 (SEQ ID NO:47) AE132 Ac-Y HArg MKLPKSAK-NH₂ 1.8 5.5 (SEQ ID NO:48)AE133 Ac-Y H MKLPKSAK-NH₂ 0.9 0.8 (SEQ ID NO:49) AE134 Ac-Y KMKLPKSAK-NH₂ 0.7 0.9 (SEQ ID NO:50) AE135 Ac-Y D MKLPKSAK-NH₂ 1.2 1.0(SEQ ID NO:51) AE136 Ac-Y E MKLPKSAK-NH₂ 0.9 0.7 (SEQ ID NO:52) AE137Ac-Y N MKLPKSAK-NH₂ 0.8 0.7 (SEQ ID NO:53) AE138 Ac-Y Q MKLPKSAK-NH₂ 0.70.8 (SEQ ID NO:54) AE139 Ac-Y F MKLPKSAK-NH₂ 0.8 1.5 (SEQ ID NO:55)AE140 Ac-Y Y MKLPKSAK-NH₂ 0.7 0.9 (SEQ ID NO:56) AE141 Ac-Y HMKLPKSAK-NH₂ 1.1 1.3 (SEQ ID NO:57) AE142 Ac-Y L MKLPKSAK-NH₂ 0.8 1.0(SEQ ID NO:58)

Table 4. Activities of substitution series at Arginine⁷⁷ in AE109 in asimultaneous assay. In this assay (as described in Example 1), theconcentration of each AE109 homolog was 64 μM. The supernatant dilutionstaken into the HT2 assays were 1:2 each allele.

In the E^(d) allele, the following four amino acids at the secondposition in AE114, where the wild-type amino acid is arginine (R),generated peptides with high activity: Arg, Cit, HArg, and Leu. The twoamino acid substitutions which resulted in peptides with least activitywere D and E. In the E^(k) system, the following three amino acids atthe second position in AE114 generated highly-active peptides: HArg,lysine, and ornithine. The two replacements in the E^(k) systemresulting in the least active peptides were D and E.

TABLE 5 Substitution Series At Methionine⁷⁸ (Position 3) in AE114.Peptide Sequence E^(d) E^(k) None 1.0 1.0 AE114 Ac-LR M KLPK-NH₂ 10.026.9 (SEQ ID NO:23) AE195 Ac-LR Orn KLPK-NH₂ 12.1 12.1 (SEQ ID NO:59)AE196 Ac-LR Cit KLPK-NH₂ 13.6 18.4 (SEQ ID NO:60) AE197 Ac-LR HArgKLPK-NH₂ 10.7 39.9 (SEQ ID NO:61) AE198 Ac-LR H KLPK-NH₂ 16.1 18.7 (SEQID NO:62) AE199 Ac-LR K KLPK-NH₂ 12.1 22.9 (SEQ ID NO:63) AE200 Ac-LR DKLPK-NH₂ 8.3 3.9 (SEQ ID NO:64) AE201 Ac-LR E KLPK-NH₂ 7.0 3.4 (SEQ IDNO:65) AE202 Ac-LR N KLPK-NH₂ 18.2 9.3 (SEQ ID NO:66) AE203 Ac-LR QKLPK-NH₂ 14.1 20.5 (SEQ ID NO:67) AE204 Ac-LR F KLPK-NH₂ 14.0 31.8 (SEQID NO:68) AE205 Ac-LR Y KLPK-NH₂ 13.9 27.4 (SEQ ID NO:69) AE206 Ac-LR LKLPK-NH₂ 11.9 33.9 (SEQ ID NO:70)

Table 5: Activities of substitution series at Methionine⁷⁸ in AE114 in asimultaneous assay. In this assay (as described in Example 1), theconcentration of the AE101 series peptide was 64 μM. The supernatantdilution taken into the HT-2 cell assay was 1:4 for each allele. TheAE101 series peptide effects in the E^(k) system were normalized to theE^(d) system by a factor of 10 since the baseline CPM for E^(k) wasapproximately 0.1 times the baseline CPM for Ed.

In the E^(d) allelic system, the following six amino acids at the thirdposition in AE114, where the wild-type amino acid is methionine (M),generated peptides with high activity: N, H, Q, F, Y, and Cit. The twoamino acid substitutions with the least activity were residues D and E.In the E^(k) system, the following seven amino acids at the thirdposition in AE114 generated highly-active peptides were: Arg, HArg, L,F, Y, M, K, and Q. The two replacements in the E^(k) system resulting inthe least active peptides were D and E.

TABLE 6 Substitution Series At Lysine⁷⁹ (Position 4) in AE114. PeptideSequence E^(d) E^(k) No peptide 1.0 1.0 AE114 Ac-LRM K LPK-NH₂ 10.6 10.7(SEQ ID NO:23) AE210 Ac-LRM Orn LPK-NH₂ 6.6 9.0 (SEQ ID NO:71) AE211Ac-LRM Cit LPK-NH₂ 1.7 3.4 (SEQ ID NO:72) AE212 Ac-LRM HArg LPK-NH₂ 13.215.4 (SEQ ID NO:73) AE213 Ac-LRM H LPK-NH₂ 6.1 5.7 (SEQ ID NO:74) AE214Ac-LRM D LPK-NH₂ 0.8 0.4 (SEQ ID NO:75) AE215 Ac-LRM E LPK-NH₂ 0.7 0.4(SEQ ID NO:76) AE216 Ac-LRM N LPK-NH₂ 2.4 3.3 (SEQ ID NO:77) AE217Ac-LRM Q LPK-NH₂ 4.0 4.4 (SEQ ID NO:78) AE218 Ac-LRM F LPK-NH₂ 3.1 9.0(SEQ ID NO:79) AE219 Ac-LRM Y LPK-NH₂ 7.1 8.8 (SEQ ID NO:80) AE220Ac-LRM M LPK-NH₂ 2.8 12.5 (SEQ ID NO:81)

Table 6. Activities of substitution series at Lysine⁷⁹ in a simultaneousassay. In this assay (as described in Example 1) the concentration ofthe AE101 series peptides was 64 μM. The supernatant dilution taken intothe HT-2 cell assay was 1:4 for each allele. The AE101 series peptideeffects in the E^(k) system were normalized to the E^(d) system throughreduction by a factor of 5, since the baseline CPM for E^(k) wasapproximately 0.2 time the baseline for Ed.

In the E^(d) allelic system, the following six amino acids at the fourthposition in AE114, where the wild-type amino acid is lysine (K),generated peptides with high activity: Met, HArg, K, Y, Orn, and H. Thetwo amino acid substitutions in peptides with the least activity were Dand E. In the E^(k) system, the following five amino acids at the fourthposition in AE114 generated peptides with high activities: HArg, M, K,Orn, and F. The two replacements in the E^(k) system resulting in theleast active peptides were D and E.

TABLE 7 Substitution Series At Leucine⁸⁰ (Position 5) in AE114. PeptideSequence E^(d) E^(k) No peptide 1.0 1.0 AE114 Ac-LRMK L PK-NH₂ 10.6 10.7(SEQ ID NO:23) AE225 Ac-LRMK Orn PK-NH₂ 10.4 13.9 (SEQ ID NO:82) AE226Ac-LRMK Cit PK-NH₂ 9.0 9.0 (SEQ ID NO:83) AE227 Ac-LRMK HArg PK-NH₂ 8.120.5 (SEQ ID NO:84) AE228 Ac-LRMK H PK-NH₂ 8.5 20.3 (SEQ ID NO:85) AE229Ac-LRMK K PK-NH₂ 13.2 16.2 (SEQ ID NO:86) AE230 Ac-LRMK D PK-NH₂ 0.6 1.1(SEQ ID NO:87) AE231 Ac-LRMK E PK-NH₂ 1.8 1.2 (SEQ ID NO:88) AE232Ac-LRMK N PK-NH₂ 12.5 8.9 (SEQ ID NO:89) AE233 Ac-LRMK Q PK-NH₂ 11.517.0 (SEQ ID NO:90) AE234 Ac-LRMK F PK-NH₂ 14.2 16.6 (SEQ ID NO:91)AE235 Ac-LRMK Y PK-NH₂ 14.6 21.8 (SEQ ID NO:92) AE236 Ac-LRMK M PK-NH₂15.2 16.9 (SEQ ID NO:93)

Table 7. Activities in substitution series at Leucine⁸⁰ in asimultaneous assay. In this assay (described in Example 1), theconcentration of the AE101 series peptides was 64 μM. The supernatantdilution taken into the HT-2 cell assay was 1:4 for each allelicsystems. The AE101 series peptide effects in the E^(k) system for 64 μMof AE peptide were normalized to the E^(d) system by a factor of 5,since the baseline CPM for E^(k) was approximately 0.2 times thebaseline CPM for E^(d).

For E^(d), the following five amino acids at the fifth position inAE114, where the wild-type amino acid is leucine (L), generated peptideswith high activity: M, Y, F, K, and N. The two amino acid substitutionswith the least activity were D and E. In the E^(k) system, the followingfive amino acids at the fifth position in AE114 generated peptides withhigh activities: Y, HArg, H, Q, and M. The two replacements in the E^(k)system with least activity were D and E.

TABLE 8 Substitution Series At Proline⁸¹ (Position 6) in AE109. PeptideSequence E^(d) E^(k) None 1.0 1.0 AE109 Ac-YRMKL P KSAK-NH₂ 16.4, 4.3,(SEQ ID NO:16) 17.6, 1.5, 7.4 1.0 AE143 Ac-YRMKL hydrP KSAK-NH₂ 19.4,4.2, (SEQ ID NO:94) 19.6, 1.5, 8.0 0.98

Table 8: Activities of hydroxyproline substitution series at Proline⁸¹.These data were generated in a simultaneous assay as described inExample 1. The concentrations of AE101 series peptides used in theseassays were 64 μM (first), 16 μM (second), and 4 μM (third) for both theE^(d) and the E^(k) systems. The supernatant dilutions taken into in theHT-2 cell assay were 1:8 for E^(d) and 1:4 for E^(k).

In both the E^(d) and E^(k) systems, replacing proline at the sixthposition of AE109 with hydroxyproline generated a peptide with equal orgreater activity than the peptide with the wild-type sequence.

TABLE 9 Substitution Series At Lysine⁸² (Position 7) in AE114. PeptideSequence E^(d) E^(k) None 1.0 1.0 AE114 Ac-LRMKLP K-NH₂ 7.7 13.2 (SEQ IDNO:23) AE240 Ac-LRMKLP Orn-NH₂ 9.8 11.5 (SEQ ID NO:95) AE241 Ac-LRMKLPCit-NH₂ 3.4 12.2 (SEQ ID NO:96) AE242 Ac-LRMKLP HArg-NH₂ 8.0 17.6 (SEQID NO:97) AE243 Ac-LRMKLP H-NH₂ 7.2 14.5 (SEQ ID NO:98) AE244 Ac-LRMKLPD-NH₂ 0.9 2.7 (SEQ ID NO:99) AE245 Ac-LRMKLP E-NH₂ 0.9 2.8 (SEQ IDNO:100) AE246 Ac-LRMKLP N-NH₂ 7.3 16.4 (SEQ ID NO:101) AE247 Ac-LRMKLPQ-NH₂ 4.5 10.9 (SEQ ID NO:102) AE248 Ac-LRMKLP F-NH₂ 17.2 16.3 (SEQ IDNO:103) AE249 Ac-LRMKLP Y-NH₂ 12.2 17.1 (SEQ ID NO:104) AE250 Ac-LRMKLPM-NH₂ 13.9 21.3 (SEQ ID NO:105) AE251 Ac-LRMKLP L-NH₂ 11.9 18.6 (SEQ IDNO:106)

Table 9: Activities of substitution series at Lysine⁸² in a simultaneousassay. In this assay (as described in Example 1), the concentration ofAE peptides was 64 μM. The supernatant dilution taken into the HT-2 cellassay was 1:4 for each allelic system. The AE101 series peptide effectin the E^(k) system for 64 μM of AE peptide was normalized to the Edsystem by a factor of 5, since the baseline CPM for E^(k) wasapproximately 0.2 times the baseline CPM for Ed.

In the E^(d) allelic system, the following five amino acids at theseventh position in AE114, where the wild-type amino acid is lysine (K),generated peptides with high activity: F, M, Y, L, and Orn. The twoamino acid substitutions which resulted in peptides with the leastactivity were D and E. In the E^(k) system, the following six aminoacids at the seventh position in AE114 generated peptides with highactivities: M, L, HArg, Y, N, and F. The two replacements in the E^(k)system with least activity were D and E.

TABLE 10 Alanine Scanning Analogs of AE101. Peptide Sequence E^(d) E^(k)None   1.0   1.0 AE120 Ac-ARMKLPKSAK-NH₂ 35.4, 18.1 36.8, 34.9 (SEQ IDNO:107) AE121 Ac-YAMKLPKSAK-NH₂  2.7, 1.9 21.6, 19.7 (SEQ ID NO:108)AE122 Ac-YRAKLPKSAK-NH₂ 16.2, 9.2 46.2, 41.4 (SEQ ID NO:109) AE123Ac-YRMALPKSAK-NH₂ 24.0, 10.7 65.0, 51.1 (SEQ ID NO:110) AE124Ac-YRMKAPKSAK-NH₂ 12.5, 7.3 66.0, 65.9 (SEQ ID NO:111) AE125Ac-YRMKLAKSAK-NH₂  2.0, 1.7 38.7, 32.8 (SEQ ID NO:112) AE126Ac-YRMKLPASAK-NH₂ 18.2, 6.8 53.4, 56.4 (SEQ ID NO:113) AE127Ac-YRMKLPKAAK-NH₂ 19.1, 12.4 63.6, 63.4 (SEQ ID NO:114) AE109Ac-YRMKLPKSAK-NH₂ 27.5, 24.3 66.0, 56.3 (SEQ ID NO:16) AE128Ac-YRMKLPKSAA-NH₂ 33.0, 29.5 51.9, 58.6 (SEQ ID NO:115)

Table 0.10: Activities of alanine substitution analogs of AE109 in asimultaneous assay. In this assay (as described in Example 1), theconcentrations of AE101 series peptides were 64 μM (first value) and 16μM (second value) for E^(d) and 64 μM (both values) for E^(k). AE109 hasthe wild-type sequence, with alanine in the ninth position. Thesupernatant dilution taken into the HT-2 cell assay was 1:2 for E^(d)and 1:2 (first value) and 1:4 (second value) for E^(k).

Substituting alanine (A) for arginine (R) at the second position and forproline (P) at the sixth position in AE109 generated peptides withsignificantly decreased enhancement in the simultaneous assay. These twopositions define two pharmacophores, i.e., side chains which arecritical for peptide activity.

Example 3 Certain D-Amino Acid Substitutions Lead to Backbone-ProtectedHomologs Retaining Partial Activity

The activity of the AE101 series peptide in the in vitro T cellfunctional assays is dependent on at least two factors: binding to thedrug's active site on MHC class II molecules and the half-life of thepeptide during the co-culture. In order to design more stable AE101series compounds, a series of analogs with single, systematic D-isomersubstitutions at each residue position was synthesized (“the D-scanseries”). Incorporating D-amino acids would presumably render suchpeptides more resistant to cleavage by proteases about the D-aminoacid-substituted residue position.

TABLE 11 D amino acid scanning analogs of AE114. Peptide Sequence E^(d)E^(k) None 1.0 1.0 AE114 Ac-LRMKLPK-NH₂ 24.1 5.2 (SEQ ID NO:23) AE160Ac-l RMKLPK-NH₂ 11.0 1.7 (SEQ ID NO:116) AE161 Ac-L r MKLPK-NH₂ 4.0 1.1(SEQ ID NO:117) AE162 Ac-LR m KLPK-NH₂ 2.2 1.3 (SEQ ID NO:118) AE163Ac-LRM k LPK-NH₂ 2.4 1.1 (SEQ ID NO:119) AE164 Ac-LRMK l PK-NH₂ 17.7 3.8(SEQ ID NO:120) AE165 Ac-LPMKL p K-NH₂ 26.6 3.6 (SEQ ID NO:121) AE166Ac-LRMKLP k-NH₂ 26.4 3.4 (SEQ ID NO:122)

Table 11: Activities of D-isomer amino acid substitution analogs ofAE114. In this assay (as described in Example 1), the concentration ofAE101 series peptides was 64 μM. Lower case letters denote D-amino acidsubstitutions. The supernatant dilutions taken into the HT-2 cell assayswere 1:8 for E^(d) and 1:4 for E^(k).

Certain homologs with individual D-isomer amino acid substitutionsretain biological activity: AE160, AE164, AE165 and AE166 for E^(d) andAE164, AE165 and AE166 for E^(k). In both alleles, the C-terminal threepositions tolerate single D-isomer substitutions better than did theC-terminal portion of the AE114 peptide.

TABLE 12 Multiple D-Isomer Substitution Analogs of AE114. PeptideSequence E^(d) E^(k) None 1.0 1.0 AE114 Ac-LRMKLPK-NH₂ 103 51.5 (SEQ IDNO:23) 115 19.3 62 4.9 5 2.1 AE170 Ac-lrmklpk-NH₂ 1.1 2.8 (SEQ ID NO:23)1.2 2.4 1.3 2.1 1.3 1.7 AE171 Ac-kplkmrl-NH₂ 1.3 3.6 (SEQ ID NO:166) 1.52.0 1.4 1.4 1.4 1.9 AE172 Ac-LRMKlpk-NH₂ 54 46.2 (SEQ ID NO:123) 7 21.12 4.2 2 2.8 AE173 Ac-LRMKLpk-NH₂ 123 51.3 (SEQ ID NO:124) 92 25.0 10 4.32 2.1

Table 12: Activities of multiple D-isomer substitution analogs of AE114in a simultaneous assay. In this assay (as described in Example 1), theconcentrations of AE101 series peptides were 64 μM, 16 μM, 4 μM and 1 μM(first through fourth values, respectively) for E^(d) and E^(k). Lowercase letters denote D-amino acid substitutions. The supernatantdilutions taken into the HT-2 cell assays were 1:2 for Ed and 1:4 forE^(k).

The all D peptide (AE170) and the “retro-inverso” all D peptide (AE171)were inactive in this assay. In the E^(k), but not E^(d) systems, Dresidues were accepted in the fifth, sixth, and seventh positions ofAE114.

Retroinverso peptides (reversed sequence, all D amino acids) sometimeshave biological activities of the natural all L amino acid peptides onwhich they are modeled. The side chain positions are comparable inretro-inverso D and all L peptides, but the backbone isproteolysis-protected. In this case, the retro-inverso all D homolog wasinactive, affirming critical steric relationships of both side chain andpeptidyl backbone interactions with the receptor. D amino acids wereacceptable in the fifth, sixth, and seventh positions, indicating thatproteolysis-resistance modifications could be introduced in this regionof the peptide without significant loss of biological activity.

Example 4 Certain N-methyl Leucine Substitutions Retain FunctionalActivity

As second peptidyl backbone modification intended to a) relationshipsalong the increase stability and b) test structure-activityrelationships along the backbone, was the substitution ofN-methyl-leucine for leucine at the first and fifth positions in AE114.

TABLE 13 N-methyl-Leucine Analogs of AE114. Peptide Sequence E^(d) E^(k)None 1.0 1.0 AE114 Ac-LRMKLPK-NH₂ 48 51.5 (SEQ ID NO:23) 65 19.3 18 4.92 2.1 AE174 Ac- (mL) RMKLPK-NH₂ 55 35.1 (SEQ ID NO:125) 40 9.8 2 3.1 11.6 AE175 Ac-LRMK (mL) PK-NH₂ 61 59.0 (SEQ ID NO:126) 36 28.5 6 6.5 12.8

Table 13: Activities of N-methyl-leucine substitution analogs of AE114in a simultaneous assay. In this assay (as described in Example 1), theconcentrations of AE101 series peptides were 64 μM, 16 μM, 4 μM and 1 μM(first through fourth values, respectively) for E^(d) and E^(k). Thesupernatant dilutions taken into the HT-2 cell assay were 1:4 for E^(d)and Ek.

N-Methyl leucine is accepted in positions one and five of AE114 withsome loss of activity in the E^(d) system. See below for the effect ofN-methyl leucine substitution for methionine in the third position.

TABLE 14 Substitution analogs of AE114. Peptide Sequence E^(d) E^(k)None 1.0 1.0 AE114 Ac-LRMKLPK-NH₂ 12.4 40.3 (SEQ ID NO:23) AE301Ac-LRLKYPK-NH₂ 12.5 59.9 (SEQ ID NO:127) AE302 Ac-LR (mL) KLPK-NH₂ 4.526.1 (SEQ ID NO:128) AE303 Ac-LR (mL) KYPK-NH₂ 4.0 25.3 (SEQ ID NO:129)AE304 Ac-LR (mL) KyPK-NH₂ 3.9 13.4 (SEQ ID NO:130) AE305 Ac-LR (mL)KYPk-NH₂ 5.2 26.8 (SEQ ID NO:131) AE306 Ac-LR (mL) KyPk-NH₂ 3.2 13.7(SEQ ID NO:132) AE307 Ac-LRLKYPk-NH₂ 13.2 56.4 (SEQ ID NO:133) AE308Ac-LRLKyPK-NH₂ 12.6 45.5 (SEQ ID NO:134) AE309 Ac-LRLKWPK-NH₂ 12.1 48.2(SEQ ID NO:135) AE235 Ac-LRMKYPK-NH₂ 12.5 56.6 (SEQ ID NO:92) AE206Ac-LRLKLPK-NH₂ 12.7 53.5 (SEQ ID NO:70) AE166 Ac-LRMKLPk-NH₂ 14.7 40.4(SEQ ID NO:121) AE164 Ac-LRMKlPK-NH₂ 12.9 43.9 (SEQ ID NO:120) AE174 Ac-(mL) RMKLPK-NH₂ 15.9 34.3 (SEQ ID NO:125) AE175 Ac-LRMK (mL) PK-NH₂ 15.958.0 (SEQ ID NO:126)

Table 14: Activities of substitution analogs of AE114 in a simultaneousassay. The data were generated in a simultaneous assay as described inExample 1. Lower case letters denote D-isomer amino acids, and (mL)denotes N-methyl leucine. The concentration of AE101 series peptides was64 μM for each allelic system. The supernatant dilutions taken into theHT-2 cell assays were 1:4 for each allelic system.

Substitution of 3-methionine by N-methyl leucine leads to a 50-70%reduction in activity (AE302 versus AE114; AE302 versus AE301). Takentogether with the results of substituting N-methyl leucine at the firstand third positions in AE174 and AE175, respectively, clearly N-methylleucine in the first (AE174), third (AE175), and fifth (AE302)positions, respectively, can be exploited to protect againstproteolysis.

Furthermore, D amino acids in the fifth position (D-leucine in AE308;D-tyrosine in AE164) and in the seventh position (D-lysine in AE166;D-lysine in AE307) also can protect against proteolysis without asignificant loss of activity.

TABLE 15 Multiple substitution analogs of AE114, targeting the E^(d)allele. Peptide Sequence E^(d) E^(k) None 1.0 1.0 AE114 Ac-LRMKLPK-NH₂228 18.2 (SEQ ID NO:23) AE340 Ac-LR Orn K HArg PK-NH₂ 46.6 39.5 (SEQ IDNO:136) AE341 Ac-LRLK HArg PK-NH₂ 38.9 42.4 (SEQ ID NO:137) AE342 Ac-LCit MKNPK-NH₂ 2.3 5.4 (SEQ ID NO:138) AE343 Ac-L Cit NKLPK-NH₂ 1.2 2.9(SEQ ID NO:139) AE344 Ac-ARNKLPK-NH₂ 7.7 2.5 (SEQ ID NO:140) AE345Ac-ARMKNPK-NH₂ 3.7 4.8 (SEQ ID NO:141) AE346 Ac-ARNKNPK-NH₂ 1.2 1.8 (SEQID NO:142) AE347 Ac-ARNKNPF-NH₂ 1.0 2.8 (SEQ ID NO:143) AE348Ac-LRNKNPF-NH₂ 13.1 6.3 (SEQ ID NO:144) AE349 Ac-LRNKNPK-NH₂ 25.3 6.1(SEQ ID NO:145) AE350 Ac-LRMKNPF-NH₂ 28.6 24.2 (SEQ ID NO:146) AE351Ac-A Cit NKNPK-NH₂ 0.8 1.6 (SEQ ID NO:147) AE235 Ac-LRMKYPK-NH₂ 29.447.7 (SEQ ID NO:92) AE120 AC-ARMKLPKSAK-NH₂ 19.4 4.8 (SEQ ID NO:107)AE131 Ac-Y Cit MKLPKSAK-NH₂ 1.6 4.1 (SEQ ID NO:47) AE195 Ac-LR OrnKLPK-NH₂ 34.3 19.8 (SEQ ID NO:59) AE202 Ac-LRNKLPK-NH₂ 34.4 5.8 (SEQ IDNO:66) AE206 Ac-LRLKLPK-NH₂ 25.5 35.3 (SEQ ID NO:70) AE227 Ac-LRMK HArgPK-NH₂ 32.3 45.6 (SEQ ID NO:84) AE232 Ac-LRMKNPK-NH₂ 24.3 15.5 (SEQ IDNO:89) AE248 Ac-LRMKLPF-NH₂ 34.7 42.6 (SEQ ID NO:103) AE301Ac-LRLKYPK-NH₂ 30.7 46.6 (SEQ ID NO:127) AE309 AC-LRLKWPK-NH₂ 13.1 38.0(SEQ ID NO:135)

Table 15: Activities of substitution analogs of AE114 in a simultaneousassay. In this assay (as described in Example 1), the concentration ofAE101 series peptides was 16 μM. The supernatant dilutions taken intothe HT-2 cell assays were 1:4.

In the above study two approaches were taken to analyze the effect ofcombinations of individual residue substitutions, each of which as asingle substitution favored E^(d) over E^(k). First, severalcombinations of two amino acid substitutions, each of which individuallyfavored Ed over E^(k), were incorporated into one, new peptide. Alanylreplacements of leucine in the first position led to a loss of activity(AE347 versus AE348; AE202 versus AE344; AE232 versus AE345). Peptidesdiffering only in methionine versus leucine in the third position werealways equally active. (AE114 versus AE206; AE235 versus AE301; AE341versus AE227). In the second approach, three or four individual favoredsubstitutions were combined together in a new peptide. Some of thesepeptides had high levels of activity.

TABLE 16 Multiple substitution analogs of AE235, targeting the E^(k)allele. Peptide Sequence E^(d) E^(k) None 1.0 1.0 AE235 Ac-LRMKYPK-NH₂14.3 8.4 (SEQ ID NO:92) AE360 Ac-L HArg MKYPK-NH₂ 5.8 2.5 (SEQ IDNO:148) AE361 Ac-L HArg LKYPK-NH₂ 8.2 4.6 (SEQ ID NO:149) AE362Ac-LKMKYPK-NH₂ 1.2 1.3 (SEQ ID NO:150) AE363 Ac-LK HArg KYPK-NH₂ 1.6 1.7(SEQ ID NO:151) AE364 Ac-LRMKYP Cit-NH₂ 14.0 16.6 (SEQ ID NO:152) AE365Ac-LR HArg MYPK-NH₂ 6.1 20.1 (SEQ ID NO:153) AE366 Ac-LR HArg KYPCit-NH₂ 7.3 49.4 (SEQ ID NO:154) AE367 Ac-LRMMYP Cit-NH₂ 1.0 6.6 (SEQ IDNO:155) AE368 Ac-LRLKYPN-NH₂ 9.4 11.2 (SEQ ID NO:156) AE301Ac-LRLKYPK-NH₂ 20.1 15.5 (SEQ ID NO:127) AE370 Ac-LRMKYPN-NH₂ 8.7 7.5(SEQ ID NO:157) AE371 Ac-FK HArg MYP Cit-NH₂ 1.6 1.5 (SEQ ID NO:158)AE114 Ac-LRMKLPK-NH₂ 15.7 1.2 (SEQ ID NO:23) AE132 Ac-Y HArgMKLPKSAK-NH₂ 3.9 2.2 (SEQ ID NO:48) AE134 Ac-YKMKLPKSAK-NH₂ 1.2 1.3 (SEQID NO:50) AE206 Ac-LRLKLPK-NH₂ 15.5 1.3 (SEQ ID NO:70) AE197 Ac-LR HArgKLPK-NH₂ 16.3 1.7 (SEQ ID NO:61) AE220 Ac-LRMMLPK-NH₂ 12.0 1.1 (SEQ IDNO:81) AE241 Ac-LRMKLP Cit-NH₂ 7.1 1.0 (SEQ ID NO:96) AE246Ac-LRMKLPN-NH₂ 21.8 1.0 (SEQ ID NO:101) AE309 Ac-LRMKWPK-NH₂ 20.0 1.8(SEQ ID NO:135)

Table 16: Simultaneous assay, substitution analogs of AE114. The datawere generated in a simultaneous assay as described in Example 1. Theconcentration of AE101 series peptides was 4 μM for each allelic system.The supernatant dilutions taken into the HT-2 cell assays were 1:8 foreach allelic system.

In the above study two approaches were taken to analyze the effect ofcombinations of residues, each of which as a single substitution favoredE^(d) over E^(k). First, several combinations of two amino acidsubstitutions, each of which individually favored E^(d) over E^(k), wereincorporated into one, new peptide. While many peptides were comparablyactive on E^(d) and E^(k), some peptides were clearly more active on oneallele than on the other. For example, AE114, AE197, AE200 and AE246 allwere more than 4 times more active on E^(d) than on E^(k). In each ofthese peptides, the fifth position was filled by leucine and the thirdposition was filled with either leucine or methionine in three peptideswith HArg occupying that position in the fourth peptide. Only AE309 hadsuch an E^(d) preference; it had a tryptophan in the fifth position. incontrast of the two peptides with greater than a 3:1 activity preferencefor E^(k) over E^(d), both had a tyrosyl residue in the fifth position(AE365, AE366) and HArg in the third position.

TABLE 17 Position 5 substitution analogs of AE114. Peptide SequenceE^(d) E^(k) None 1.0 1.0 AE114 Ac-LRMKLPK-NH₂ 47 2.8 (SEQ ID NO:23)AE235 Ac-LRMKYPK-NH₂ 51 26.8 (SEQ ID NO:92) AE322 Ac-LRMK (X2) PK-NH₂ 373.0 (SEQ ID NO:159) AE323 Ac-LRMK (X3) PK-NH₂ 53 8.4 (SEQ ID NO:159)AE324 Ac-LRMK (X4) PK-NH₂ 25 3.1 (SEQ ID NO:159) AE325 Ac-LRMK (X5)PK-NH₂ 49 4.7 (SEQ ID NO:159) AE326 Ac-LRMK (X6) PK-NH₂ 38 9.6 (SEQ IDNO:159) AE327 Ac-LRMK (X8) PK-NH₂ 10 2.2 (SEQ ID NO:159) AE328 Ac-LRMK(X9) PK-NH₂ 4.5 2.0 (SEQ ID NO:159) AE329 Ac-LRMK (X12) PK-NH₂ 35 2.8(SEQ ID NO:159) AE330 Ac-LRMK (X13) PK-NH₂ 32 2.6 (SEQ ID NO:159) AE331Ac-LRMK (X14) PK-NH₂ 24 12.2 (SEQ ID NO:159) AE332 Ac-LRMK (X15) PK-NH₂29 26.4 (SEQ ID NO:159)

Table 17: Activities of position 5 substitution analogs of AE114 in asimultaneous assay. In this assay (as described in Example 1), theconcentration of AE101 series peptides was 4 μM for E^(d) and 64 μM forE^(k). The supernatant dilutions taken in the HT-2 cell assays were −1:4for E^(d) and 1:4 for E^(k). The following side chain structures weresubstituted at position 5: X2=p-chloro-Phe; X3=p-fluoro-Phe;X4=p-nitro-Phe; X5=α-amino-4-phenylbutyrate; X6=β-thienylalanine (Thi);X8=di-bromo-tyrosine; X9=di-iodo-tyrosine; X12=β-1-napthyl-alanine;X13=β-2-napthyl-alanine; X14=1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid (Tic); andX15=1,2,3,4-tetrahydroisoquinoline-7-hydroxy-3-carboxylic acid[Tic(OH)].

The Tic(OH) substitution (AE332) is as potent on E^(k) as is the5-tyrosyl reference peptide AE235. The Tic(OH) residue can besuperimposed on the tyrosyl structure, with the addition of a methylenebridge between the 2-phenyl carbon and the imido nitrogen in thepeptidyl bond of that residue. That bridge mimics proline. Lack of thedistal phenolic hydroxyl (AE332 versus AE331) lessens activity. ThisAE332 Tic(OH) homolog while not significantly more potent than the AE235tyrosyl homolog is nevertheless much more resistant to proteolysis andcan therefor be expected to be considerably more potent in vivo.

TABLE 18 Cyclical analogs of AE-114. Peptide Sequence E^(d) E^(k) None1.0 1.0 AE114 Ac-LRMKLPK-NH₂ 12.4 40.3 (SEQ ID NO:23) AE381cyclic LRMKLPK 2.1 0.56 (SEQ ID NO:23) AE382 Ac-LRMKLPK 1.9 18.1 (SEQ IDNO:23) AE235 Ac-LRMKYPK-NH₂ 12.5 56.6 (SEQ ID NO:92)

Table 18: Activities of cyclical analogs of AE114 in a simultaneousassay. In this assay (as described in Example 1), the concentration ofthe AE101 series peptides was 64 μM. The supernatant dilution taken intothe HT-2 cell assays was 1:4 for each allelic system. The AE381 peptideis a “head-to-tail” cyclization from the amino-terminus to thecarboxyl-terminus of AE114. The AE382 peptide is a “side-to-tail”cyclization of AE114 from the epsilon-amino group of the 4-lysyl residueto the C-terminal carboxyl group, retaining the amino-terminal acetylgroup.

The “head to tail” cyclical peptide AE381 is weak in simultaneous assayson E^(d) and E^(k). The “side to tail” cyclical peptide AE382 ismoderately active on E^(k) and relatively inactive on E^(d). Theseresults contrast to the potent immunosuppressive activities of thesepeptides in the antigenic peptide prepulse assay (Example 5, Table 18)and the processed antigen assay (Example 6, Table 34).

Example 5 Effect of AE109 and AE114 Substitutions on the “PeptidePrepulse Assay”

TABLE 19 C-terminal Truncation Analogs of AE101. Peptide Sequence E^(d)E^(k) None 1.00 1.00 AE109 Ac-YRMKLPKSAK-NH₂ 0.11 0.07 (SEQ ID NO:16)AE167 Ac-LRMKLPKPPP-NH₂ 0.23 0.01 (SEQ ID NO:18) AE168 Ac-LRMKLPKPPK-NH₂0.10 0.01 (SEQ ID NO:19) AE113 Ac-YRMKLPK-NH₂ 0.10 0.04 (SEQ ID NO:22)AE115 Ac-YRMKLP-NH₂ 0.49 0.07 (SEQ ID NO:24) AE116 Ac-YRMKL-NH₂ 0.800.10 (SEQ ID NO:25) AE1l7 Ac-YRMK-NH₂ 0.65 0.09 (SEQ ID NO:26) AE118Ac-YRM-NH₂ 0.68 0.35 (SEQ ID NO:27)

Table 19: Activities of C-terminal truncation analogs of AE101 in anantigenic peptide prepulse assay. These data were generated in antigenicpeptide prepulse assays as described in Example 1 with the followingmodifications. Paraformaldehyde-treated APC (fixed APC) expressing MHCclass II molecules were incubated with antigenic peptide for 6 h(HEL106-116 for E^(d) and PGCC81-104 for E^(k)). After this incubation,the prepulsed APC were washed and cultured with T hybridoma cells andAE101 series peptides. The antigenic peptide concentration during theprepulse was 12 μM for Ed and 20 μM for E^(k). The AE101 series peptideconcentrations were 64 μM for E^(d) and 16 μM for E^(k). The dilutionsof supernatant taken into the HT-2 cell assay were 1:4 for Ed and 1:2for E^(k).

The results in the E^(d) allele differed significantly from results inthe E^(k) allele in both this study of the effects of C-terminaltruncations of AE113 in the peptide prepulse assay and in thesimultaneous assay (Example 1, Table 1). While, in the E^(d) system,loss of one C-terminal residue (AE115) decreased activity relative toAE113 by half, peptides as short as four amino acids (AE117) retainedfull activity in both this peptide prepulse assay and in thesimultaneous assay (Example 1, Table 1).

TABLE 20 Substitution Series At Arginine⁷⁷ (Position 2) in AE109.Peptide Sequence E^(d) E^(k) None 1.00 1.00 AE101 YRMKLPKSAKPVSQMR 0.250.89 (SEQ ID NO:3) AE109 Ac-Y R MKLPKSAK-NH₂ 0.28 0.28 (SEQ ID NO:16)AE121 Ac-Y A MKLPKSAK-NH₂ 0.53 0.59 (SEQ ID NO:45) AE130 Ac-Y OrnMKLPKSAK-NH₂ 0.79 0.66 (SEQ ID NO:46) AE131 Ac-Y Cit MKLPKSAK-NH₂ 0.690.71 (SEQ ID NO:47) AE132 Ac-Y Harg MKLPKSAK-NH₂ 0.58 0.1 (SEQ ID NO:48)AE133 Ac-Y H MKLPKSAK-NH₂ 0.39 0.86 (SEQ ID NO:49) AE134 Ac-Y KMKLPKSAK-NH₂ 0.60 0.43 (SEQ ID NO:50) AE135 Ac-Y D MKLPKSAK-NH₂ 0.700.95 (SEQ ID NO:51) AE136 Ac-Y E MKLPKSAK-NH₂ 0.54 0.97 (SEQ ID NO:52)AE137 Ac-Y N MKLPKSAK-NH₂ 0.57 0.72 (SEQ ID NO:53) AE138 Ac-Y QMKLPKSAK-NH₂ 0.73 0.85 (SEQ ID NO:54) AE139 Ac-Y F MKLPKSAK-NH₂ 0.670.38 (SEQ ID NO:55) AE140 Ac-Y Y MKLPKSAK-NH₂ 0.78 0.79 (SEQ ID NO:56)AE141 Ac-Y M MKLPKSAK-NH₂ 0.41 0.52 (SEQ ID NO:57) AE142 Ac-Y LMKLPKSAK-NH₂ 0.86 0.46 (SEQ ID NO:58)

Table 20: Activities of substitution series at Arginine⁷⁷ in AE109 in anantigenic peptide prepulse assay. These data were generated in antigenicpeptide prepulse assays carried out as described in Example 1 with thefollowing modifications. The antigenic peptide concentrations during theprepulse was 3 μM for E^(d) and 20 μM for E^(k). The AE peptideconcentrations were 64 μM for E^(d) and 16 μM for E^(k). The supernatantdilutions taken into the HT-2 cell assay were 1:2 for E and 1:2 forE^(k).

The R⁷⁷ (position 2) substituted peptides with the greatest activitiesin the simultaneous assay (Example 1, Table 4) had the greatest activityin the peptide prepulse assay reported here. This mirror imaging ofactivities in these two assays supports a conclusion of validity aboutthe structure/activity relationships demonstrated in these experimentstesting the effects of amino acid replacements at each residue position.

TABLE 21 Substitution series at Proline⁸¹ (Position 5) in AE109. PeptideSequence E^(d) E^(k) None 1.00 1.00 AE109 Ac-YRMKL P KSAK-NH₂ 0.10 0.07(SEQ ID NO:16) AE143 Ac-YRMKL hydrP KSAK-NH₂ 0.18 0.10 (SEQ ID NO:94)

Table 21: Activities of hydroxyproline substitution at Proline⁸¹ in anantigenic peptide prepulse assay. In these assays (as described inExample 1), the concentrations of antigenic peptide in the antigenprepulse were 24 μM for Ed and 20 μM for E^(k). The concentrations of AEpeptides were 64 μM for E^(d) and 16 μM for E^(k). The supernatantdilutions taken into the HT-2 cell assay were 1:8 for E^(d) and 1:2 forE^(k).

Hydroxyproline at position 5 in AE109 was as potent as proline in thesimultaneous assay (Example 1, Table 8) and in the peptide prepulseassay shown here. This result is generally interpreted to mean thatthere is not a crucial structural interaction between the aliphatic ringof proline and the protein receptor, but instead that the role of theproline is primarily to stabilize local configuration. Since the Tic(OH)structure (an analog of proline) is active, the adjacent placement ofproline like Tic(OH) (in the fifth position) to proline in the sixthposition creates a type II polyprolyl helix configuration through thatregion.

TABLE 22 D amino acid scan analogs of AE114. Peptide Sequence E^(d)E^(k) None 1.00 1.00 AE114 Ac-LRMKLPK-NH₂ 0.02 0.15 (SEQ ID NO:23) AE160Ac-1 RMKLPK-NH₂ 0.40 0.47 (SEQ ID NO:116) AE161 Ac-L r MKLPK-NH₂ 0.300.85 (SEQ ID NO:117) AE162 Ac-LR m KLPK-NH₂ 0.28 0.61 (SEQ ID NO:118)AE163 Ac-LRM k LPK-NH₂ 0.39 0.99 (SEQ ID NO:119) AE164 Ac-LRMK 1 PK-NH₂0.37 0.21 (SEQ ID NO:120) AE165 Ac-LRMKL p K-NH₂ 0.25 0.22 (SEQ IDNO:121) AE166 Ac-LRMKLP k-NH₂ 0.11 0.15 (SEQ ID NO:122)

Table. 22: Activities of D-amino acid substitutions in AE114 in anantigenic peptide prepulse assay. In this assay (as described in Example1), the concentrations of antigenic peptides in the prepulse were 6 μMfor E^(d) and 20 μM for E^(k). The concentrations of AE101 seriespeptides were 64 μM for Ed and 16 μM for E^(k). The supernatantdilutions taken into the HT-2 cell assay were 1:8 for E^(d) and 1:2 forE^(k).

The activities of AE114 homologs with individual D-amino acidsubstitutions at each residue position had broadly comparable activitiesin the simultaneous assay (Example 3, Table 11) and in the peptideprepulse assay reported here. In particular, D-amino acid replacementsin residue positions 2 through 5 significantly decreased activity.

TABLE 23 N-methyl-Leucine Analogs of AE114. Peptide Sequence E^(d) E^(k)None 1 1 AE114 Ac-LRMKLPK-NH₂ 0.41 0.78 (SEQ ID NO:23) AE174Ac-(mL)RMKLPK-NH₂ 0.6 0.81 (SEQ ID NO:125) AE175 Ac-LRMK(mL)PK-NH₂ 0.70.87 (SEQ ID NO:126)

Table 23: Activities of N-methyl-leucine analogs of AE114 in anantigenic peptide prepulse assay. In this assay (as described in Example1), the concentrations of antigenic peptides during the prepulse were 64μM for E^(d) and 64 μM for Ek. The concentrations of AE101 seriespeptides used were 1:4 μM for E^(d) and 1:8 μM for E^(k). Thesupernatant dilutions taken into the HT-2 cell assay were 1:8 for E^(d)and 1:2 for E^(k).

As in the simultaneous assay with these N-methyl-leucine substitutions(Example 4, Table 13), N-methyl-leucine substitutions had a small degreeof activity loss relative to the control peptide AE114. Suchsubstitutions might be expected in vivo to lead to increased potency dueto proteolysis protection of these substrates. Table 24: Substitutionanalogs of AE-114.

TABLE 24 Substitution analogs of AE-114. Peptide Sequence E^(d) E^(k)None 1.00 1.00 AE114 Ac-LRMKLPK-NH₂ 0.41 0.78 (SEQ ID NO:23) AE301Ac-LRLKYPK-NH₂ 0.48 0.80 (SEQ ID NO:127) AE302 Ac-LR (mL) KLPK-NH₂ 0.820.85 (SEQ ID NO:128) AE303 Ac-LR (mL) KYPK-NH₂ 0.78 1.00 (SEQ ID NO:129)AE304 Ac-LR (mL) KyPK-NH₂ 0.78 0.76 (SEQ ID NO:130) AE305 Ac-LR (mL)KYPk-NH₂ 0.72 0.91 (SEQ ID NO:131) AE306 Ac-LR (mL) KyPk-NH₂ 0.68 1.00(SEQ ID NO:132) AE307 Ac-LRLKYPk-NH₂ 0.59 1.10 (SEQ ID NO:133) AE308Ac-LRLKyPK-NH₂ 0.93 0.63 (SEQ ID NO:134) AE309 Ac-LRLKWPK-NH₂ 0.44 0.81(SEQ ID NO:135) AE235 Ac-LRMKYPK-NH₂ 0.30 0.80 (SEQ ID NO:92) AE206Ac-LRLKLPK-NH₂ 0.52 1.00 (SEQ ID NO:70) AE166 Ac-LRMKLPk-NH₂ 0.65 0.83(SEQ ID NO:122) AE164 Ac-LRMKlPK-NH₂ 0.59 0.67 (SEQ ID NO:120) AE174 Ac-(mL) RMKLPK-NH₂ 0.60 0.81 (SEQ ID NO:125) AE175 Ac-LRMK (mL) PK-NH₂ 0.700.87 (SEQ ID NO:126)

Table 24: Activities of substitution analogs of AE114 in an antigenicpeptide prepulse assay. In this assay (as described in Example 1), theconcentrations of antigenic peptides during the prepulse were 24 μM forE^(d) and 0.625 μM for E^(k). The concentrations of AE101 seriespeptides used were 64 μM for E^(d) and 64 μM for E^(k). The supernatantdilutions taken into the HT-2 cell assay were 1:4 for E^(d) and 1:8 forE^(k).

The results in this assay constitute, relatively, a mirror image of theresult in the simultaneous assay (Table 14). Substitutions of Met³ byN-methyl-leucine led to a loss of activity compared to AE114.Furthermore, D amino acids in the fifth position (D-leucine in AE308;D-tyrosine in AE164) and in the seventh position (D-lysine in AE166;D-lysine in AE307) also can protect against proteolysis without asignificant loss of activity.

TABLE 25 Multiple substitution analogs of AE-114. targeting theE^(d)allele. Peptide Sequence E^(d) E^(k) None 1.00 1.00 AE114Ac-LRMKLPK-NH₂ 0.31 0.79 (SEQ ID NO:23) AE340 Ac-LR Orn K Harg PK-NH₂0.10 0.83 (SEQ ID NO:136) AE341 Ac-LRLK Harg PK-NH₂ 0.12 1.10 (SEQ IDNO:137) AE342 Ac-L Cit MKNPK-NH₂ 0.67 1.10 (SEQ ID NO:138) AE343 Ac-LCit NKLPK-NH₂ 0.55 0.90 (SEQ ID NO:139) AE344 Ac-ARNKLPK-NH₂ 0.46 0.76(SEQ ID NO:140) AE345 Ac-ARMKNPK-NH₂ 0.61 0.86 (SEQ ID NO:141) AE346Ac-ARNKNPK-NH₂ 0.65 0.77 (SEQ ID NO:142) AE347 Ac-ARNKNPF-NH₂ 0.64 1.10(SEQ ID NO:143) AE348 Ac-LRNKNPF-NH₂ 0.39 0.72 (SEQ ID NO:144) AE349Ac-LRNKNPK-NH₂ 0.69 0.81 (SEQ ID NO:145) AE350 Ac-LRMKNPF-NH₂ 0.82 0.86(SEQ ID NO:146) AE351 Ac-A Cit NKNPK-NH₂ 0.42 0.90 (SEQ ID NO:147) AE235Ac-LRMKYPK-NH₂ 0.25 0.76 (SEQ ID NO:92) AE120 Ac-ARMKLPKSAK-NH₂ 0.630.62 AE131 Ac-Y Cit MKLPKSAK-NH₂ 0.61 0.98 (SEQ ID NO:47) AE195 Ac-LRORN KLPK-NH₂ 0.41 0.74 (SEQ ID NO:59) AE202 Ac-LRNKLPK-NH₂ 0.30 0.84(SEQ ID NO:66) AE206 Ac-LRLKLPK-NH₂ 0.18 0.68 (SEQ ID NO:70) AE227Ac-LRMK Harg PK-NH₂ 0.28 0.87 (SEQ ID NO:84) AE232 Ac-LRMKNPK-NH₂ 0.440.92 (SEQ ID NO:89) AE248 Ac-LRMKLPF-NH₂ 0.63 0.98 (SEQ ID NO:103) AE301Ac-LRLKYPK-NH₂ 0.66 0.86 (SEQ ID NO:127) AE309 Ac-LRLKWPK-NH₂ 0.48 0.93(SEQ ID NO:135)

Table 25: Activities of multiple substitution analogs of AE114,targeting the E^(d) allele, in the antigenic peptide prepulse assay. Inthis assay (as described in Example 1), the concentrations of antigenicpeptides during the prepulse were 24 μM for E^(d) and 2.5 μM for E^(k).The concentrations of AE101 series peptides used were 64 μM for E^(d)and 64 μM for Ek. The supernatant dilutions taken into the HT-2 cellassay were 1:4 for E^(d) and 1:10 for E^(k).

The results in these assays parallel the results in the simultaneousassay (Example 4, Table 15).

TABLE 26 Multiple substitution analogs of AE235, targeting the E^(k)allele. Peptide Sequence E^(d) E^(k) None 1.0 1.0 AE235 Ac-LRMKYPK-NH₂0.23 0.60 (SEQ ID NO:92) AE360 Ac-L Harg MKYPK-NH₂ 0.91 0.94 (SEQ IDNO:148) AE361 Ac-L Harg LKYPK-NH₂ 0.83 1.00 (SEQ ID NO:149) AE362Ac-LKMKYPK-NH₂ 0.76 0.91 (SEQ ID NO:150) AE363 Ac-LK Harg KYPK-NH₂ 0.600.97 (SEQ ID NO:151) AE364 Ac-LRMKYP Cit-NH₂ 0.34 0.93 (SEQ ID NO:152)AE365 Ac-LR Harg MYPK-NH₂ 0.70 0.42 (SEQ ID NO:153) AE366 Ac-LR Harg KYPCit-NH₂ 0.44 0.94 (SEQ ID NO:154) AE367 Ac-LRMMYP Cit-NH₂ 1.10 0.99 (SEQID NO:155) AE368 Ac-LRLKYPN-NH₂ 0.55 0.92 (SEQ ID NO:156) AE301Ac-LRLKYPK-NH₂ 0.34 0.99 (SEQ ID NO:127) AE370 Ac-LRMKYPN-NH₂ 0.74 0.58(SEQ ID NO:157) AE371 Ac-FK Harg MYP Cit-NH₂ 0.90 0.99 (SEQ ID NO:158)AE114 Ac-LRMKLPK-NH₂ 0.28 0.98 (SEQ ID NO:23) AE132 Ac-Y HargMKLPKSAK-NH₂ 0.82 0.89 (SEQ ID NO:48) AE134 Ac-YKMKLPKSAK-NH₂ 0.83 0.98(SEQ ID NO:50) AE206 Ac-LRLKLPK-NH₂ 0.38 0.96 (SEQ ID NO:70) AE197 Ac-LRHarg KLPK-NH₂ 0.32 0.96 (SEQ ID NO:61) AE220 Ac-LRMMLPK-NH₂ 0.73 0.92(SEQ ID NO:81) AE241 Ac-LRMKLP Cit-NH₂ 0.70 0.99 (SEQ ID NO:96) AE246Ac-LRNKLPN-NH₂ 0.69 0.98 (SEQ ID NO:101) AE309 Ac-LRMKWPK-NH₂ 0.25 0.98(SEQ ID NO:135)

Table 26: Activities of multiple substitution analogs of AE235,targeting the E^(k) allele, in an antigenic peptide prepulse assay. Inthis assay (as described in Example 1), the concentrations of antigenicpeptides during the prepulse were 24 μM for E^(d) and 1.25 μM for E^(k).The concentrations of AE101 series peptides used were 64 μM for E^(d)and 64 μM for Ek. The supernatant dilutions taken into the HT-2 cellassay were 1:2 for E^(d) and 1:8 for E^(k).

Activities of individual AE101 series peptides in this assay paralleledtheir level of activity in the simultaneous assay (Example 4, Table 15).

TABLE 27 Position 5 substitution analogs of AE114. Peptide SequenceE^(d) E^(k) None 1.0 1.0 AE114 Ac-LRMKLPK-NH₂ 0.32 0.69 (SEQ ID NO:23)AE235 Ac-LRMKYPK-NH₂ 0.40 0.83 (SEQ ID NO:92) AE322 Ac-LRMK (X2) PK-NH₂0.42 0.64 (SEQ ID NO:159) AE323 Ac-LRMK (X3) PK-NH₂ 0.19 0.50 (SEQ IDNO:159) AE324 Ac-LRMK (X4) PK-NH₂ 0.44 0.68 (SEQ ID NO:159) AE325Ac-LRMK (X5) PK-NH₂ 0.18 0.58 (SEQ ID NO:159) AE326 Ac-LRMK (X6) PK-NH₂0.40 0.52 (SEQ ID NO:159) AE327 Ac-LRMK (X8) PK-NH₂ 0.74 0.99 (SEQ IDNO:159) AE328 Ac-LRMK (X9) PK-NH₂ 0.71 0.74 (SEQ ID NO:159) AE329Ac-LRMK (X12) PK-NH₂ 0.34 0.40 (SEQ ID NO:159) AE330 Ac-LRMK (X13)PK-NH₂ 0.25 0.37 (SEQ ID NO:159) AE331 Ac-LRMK (X14) PK-NH₂ 0.40 0.84(SEQ ID NO:159) AE332 Ac-LRMK (X15) PK-NH₂ 0.33 0.61 (SEQ ID NO:159)

Table 27: Activities of position 5 substitution analogs in an antigenicpeptide prepulse assay. In this assay (as described in Example 1), theconcentrations of antigenic peptides during the prepulse were 24 μM forE^(d) and 1.25 μM for E^(k). The concentrations of AE101 series peptidesused were 64 μM for E^(d) and 64 μM for E^(k). The supernatant dilutionstaken into the HT-2 cell assay was 1:8 for E^(d) and 1:8 for E^(k). Thefollowing side chain structures were substituted at position 5:X2=p-chloro-Phe; X3=p-fluoro-Phe; X4=p-nitro-Phe;X5=α-amino-4-phenylbutyrate; X6=β-thienylalanine (Thi);X8=di-bromo-tyrosine; X9=di-iodo-tyrosine; X12=β-1-napthyl-alanine;X13=β-2-napthyl-alanine; X14=1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid (Tic); andX15=1,2,3,4-tetrahydroisoquinoline-7-hydroxy-3-carboxylic acid[Tic(OH)].

Activities of individual AE101 series peptides in this assay paralleledtheir level of activity in the simultaneous assay (Example 4, Table 17).

TABLE 28 Cyclical analogs of AE-114. Peptide Sequence E^(d) E^(k) None1.0 1.0 AE114 Ac-LRMKLPK-NH₂ 0.41 0.78 (SEQ ID NO:23) AE381Ac-LRMKLPK-NH₂ 0.19 0.28 (SEQ ID NO:23) AE382 Ac-LRMKLPK-NH₂ 0.52 0.70(SEQ ID NO:23) AE235 Ac-LRMKYPK-NH₂ 0.30 0.80 (SEQ ID NO:92)

Table 28: Activities of multiple substitution analogs of AE235,targeting the E^(k) allele, in an antigenic peptide prepulse assay. Inthis assay (as described in Example 1), the concentrations of antigenicpeptides during the prepulse were 24 μM for E^(d) and 1.25 μM for E^(k).The concentrations of AE101 series peptides used were 64 μM for E^(d)and E^(k). The supernatant dilutions taken into the HT-2 cell assay were1:4 for E^(d) and E^(k).

In sharp contrast to the pattern with all linear AE101 series peptides,wherein the activity in the antigenic prepulse assay was the mirrorimage of activity in the simultaneous assay, the “head-to-tail” cyclicalpeptide AE381 was significantly more active in the antigenic peptideprepulse assay than in the simultaneous assay. This finding isconsistent with the hypothesis that this cyclic peptide binds verytightly to the allosteric effector site in a fashion which did notpermit entry of a second antigenic peptide in to the antigenic peptidebinding site of the MHC class II molecules.

Example 6 Effects on the “Processed Antigen” Assay

The “processed antigen assay” was carried out under essentially the sameconditions as the “peptide prepulse” assay, with the followingmodifications. Untreated APC were incubated at 1×10⁶/mL in 24-wellplates (1 mL/well) with native protein antigen for 8 h. Followingincubation, the pulsed APC were washed, treated with mitomycin C, andwere washed again. The assay conditions were then as described for“peptide prepulse” above. The baseline T cell response was measured byculturing T hybridoma cells with the native antigen-prepulsed APC in theabsence of AE101 peptides.

TABLE 29 Leucine⁸⁰ analogs of AE114. 20 μM 10 μM 5 μM HEL HEL HELPeptide Sequence E^(d) E^(d) E^(d) None 1.0,1.0 1.0,1.0 1.0,1.0 AE114Ac-LRMK L PK-NH₂ 0.53, 0.29, 0.23, (SEQ ID NO:23) 0.44 0.28 0.37 AE225Ac-LRMK Orn PK-NH₂ 0.59, 0.32, 0.26, (SEQ ID NO:82) 0.48 0.30 0.38 AE226Ac-LRMK Cit PK-NH₂ 0.95, 0.68, 0.60, (SEQ ID NO:83) 0.87 0.58 0.34 AE227Ac-LRMK HargPK-NH₂ 0.45, 0.18, 0.18, (SEQ ID NO:84) 0.36 0.19 0.33 AE228Ac-LRMK H PK-NH₂ 0.71, 0.41, 0.35, (SEQ ID NO:85) 0.61 0.41 0.45 AE229Ac-LRMK K PK-NH₂ 0.71, 0.55, 0.38, (SEQ ID NO:86) 0.62 0.49 0.58 AE230Ac-LRMK D PK-NH₂ 1.1, 0.93, 0.81, (SEQ ID NO:87) 0.98 0.77 0.62 AE231Ac-LRMK E PK-NH₂ 1.0, 1.1 0.94, 0.96, (SEQ ID NO:88) 0.83 0.85 AE232Ac-LRMK N PK-NH₂ 0.97, 0.68, 0.50, (SEQ ID NO:89) 0.83 0.61 0.50 AE233Ac-LRMK Q PK-NH₂ 0.74, 0.53, 0.48, (SEQ ID NO:90) 0.68 0.47 0.36 AE234Ac-LRMK F PK-NH₂ 0.98, 0.65, 0.51, (SEQ ID NO:91) 0.88 0.60 0.62 AE235Ac-LRMK Y PK-NH₂ 0.71, 0.42, 0.33, (SEQ ID NO:92) 0.56 0.39 0.38 AE236Ac-LRMK M PK-NH₂ 0.76, 0.56, 0.41, (SEQ ID NO:93) 0.72 0.53 0.58

Table 2.9: Activities of substitution series at Leucine⁸⁰ in AE114 in aprocessed antigen assay. These data presented were generated asdescribed in the legend of Table 14, with the following modifications.The results in this assay constitute, relatively, a mirror image of theresult in the simultaneous assay (Example 1, Table 7) and parallel thoseof the antigenic peptide prepulse assay (Example 5, Table 24). UntreatedAPC were incubated with native HEL, instead of antigenic peptide, for 8h. After incubation, the pulsed cells were washed and mitomycin Ctreated before being cocultured with AE-peptides and T cell hybridomas.Wells containing only T cells and native HEL-prepulsed APC were used todetermine the Baseline response, or 1, in the absence of AE peptides.The concentration of AE peptide used was 64 μM for these assays. Thesupernatant dilutions used in the HT-2 cell assay were 1:4 (first value)and 1:8 (second value).

AE114 homologs with various amino acid substitutions in the fifthposition which were most potent in the simultaneous assay (Example 1,Table 7) and in a peptide prepulse assay (Example 5, Table 15) were mostactive in this processed antigen assay.

TABLE 30 Substitution analogs of AE-114, Peptide Sequence E^(d) None 1.0AE114 Ac-LRMKLPK-NH₂ 0.22 (SEQ ID NO:23) AE301 Ac-LRLKYPK-NH₂ 0.19 (SEQID NO:127) AE302 Ac-LR (mL) KLPK-NH₂ 0.51 (SEQ ID NO:128) AE303 Ac-LR(mL) KYPK-NH₂ 0.82 (SEQ ID NO:129) AE304 Ac-LR (mL) KyPK-NH₂ 0.77 (SEQID NO:130) AE305 Ac-LR (mL) KYPk-NH₂ 0.70 (SEQ ID NO:131) AE306 Ac-LR(mL) KyPk-NH₂ 0.68 (SEQ ID NO:132) AE307 Ac-LRLKYPk-NH₂ 0.48 (SEQ IDNO:133) AE308 Ac-LRLKyPK-NH₂ 0.60 (SEQ ID NO:134) AE309 Ac-LRLKWPK-NH₂0.29 (SEQ ID NO:135) AE235 Ac-LRMKYPK-NH₂ 0.19 (SEQ ID NO:92) AE206Ac-LRLKLPK-NH₂ 0.22 (SEQ ID NO:70) AE166 Ac-LRMKLPk-NH₂ 0.36 (SEQ IDNO:122) AE164 Ac-LRMKlPK-NH₂ 0.64 (SEQ ID NO:120) AE174 Ac-(mL)RMKLPK-NH₂ 0.86 (SEQ ID NO:125) AE175 Ac-LRMK (mL) PK-NH₂ 0.70 (SEQ IDNO:126)

Table 30: Activities of substitution analogs of AE114 in a processedantigen assay. In this assay (as described in the legend of Table 30)the concentration of native HEL used during the prepulse was 10 μM. Theconcentration of AE peptide used was 64 μM for this assay. Thesupernatant dilution used in the HT-2 cell assay was 1:8.

The results in this assay constitute, relatively, a mirror image of theresult in the simultaneous assay (Example 1, Table 14) and parallelthose of the antigenic peptide prepulse assay (Example 5, Table 24).Substitutions of Met³ by N-methyl-leucine led to a loss of activitycompared to AE114. Furthermore, D amino acids in the fifth position(D-leucine in AE308; D-tyrosine in AE164) and in the seventh position(D-lysine in AE166; D-lysine in AE307) also can protect againstproteolysis without a significant loss of activity.

TABLE 31 Multiple substitution analogs of AE114, targeting the E^(d)allele. Peptide Sequence E^(d) None 1.00 peptide AE114 Ac-LRMKLPK-NH₂0.49 (SEQ ID NO:23) AE340 Ac-LR Orn K Harg PK-NH₂ 0.16 (SEQ ID NO:136)AE341 Ac-LRLK Harg PK-NH₂ 0.12 (SEQ ID NO:137) AE342 Ac-L Cit MKNPK-NH₂0.64 (SEQ ID NO:138) AE343 Ac-L Cit NKLPK-NH₂ 0.69 (SEQ ID NO:139) AE344Ac-ARNKLPK-NH₂ 0.76 (SEQ ID NO:140) AE345 Ac-ARMKNPK-NH₂ 0.81 (SEQ IDNO:141) AE346 Ac-ARNKNPK-NH₂ 0.78 (SEQ ID NO:142) AE347 Ac-ARNKNPF-NH₂0.80 (SEQ ID NO:143) AE348 Ac-LRNKNPF-NH₂ 0.81 (SEQ ID NO:144) AE349Ac-LRNKNPK-NH₂ 0.64 (SEQ ID NO:145) AE350 Ac-LRMKNPF-NH₂ 0.56 (SEQ IDNO:146) AE351 Ac-A Cit NKNPK-NH₂ 0.87 (SEQ ID NO:147) AE235Ac-LRMKYPK-NH₂ 0.53 (SEQ ID NO:92) AE120 Ac-ARMKLPKSAK-NH₂ 0.51 (SEQ IDNO:107) AE131 Ac-Y Cit MKLPKSAK-NH₂ 0.49 (SEQ ID NO:47) AE195 Ac-LR OrnKLPK-NH₂ 0.31 (SEQ ID NO:59) AE202 Ac-LRNKLPK-NH₂ 0.76 (SEQ ID NO:66)AE206 Ac-LRLKLPK-NH₂ 0.41 (SEQ ID NO:70) AE227 Ac-LRMK Harg PK-NH₂ 0.35(SEQ ID NO:84) AE232 Ac-LRMKNPK-NH₂ 0.46 (SEQ ID NO:89) AE248Ac-LRMKLPF-NH₂ 0.30 (SEQ ID NO:103) AE301 Ac-LRLKYPK-NH₂ 0.46 (SEQ IDNO:127) AE309 Ac-LRLKWPK-NH₂ 0.44 (SEQ ID NO:135)

Table 31: Activities of multiple substitution analogs of AE114,targeting the E^(d) allele, in a processed antigen assay. In this assay(as described in the legend of Table 30), the concentration of nativeHEL used during the prepulse was 10 μM. The concentration of AE peptideused was 64 μM for this assay. The supernatant dilution used in the HT-2cell assay was 1:4.

The results in this assay constitute, relatively, a mirror image of theresult in the simultaneous assay (Example 1, Table 15) and parallelthose of the antigenic peptide prepulse assay (Example 5, Table 25).

TABLE 32 Multiple substitution analogs of AE235, targeting the E^(k)allele. Peptide Sequence E^(d) None 1.0 AE235 Ac-LRMKYPK-NH₂ 0.46 (SEQID NO:92) AE360 Ac-L Harg MKYPK-NH₂ 0.64 (SEQ ID NO:148) AE361 Ac-L HargLKYPK-NH₂ 0.51 (SEQ ID NO:149) AE362 Ac-LKMKYPK-NH₂ 0.58 (SEQ ID NO:150)AE363 Ac-LK Harg KYPK-NH₂ 0.75 (SEQ ID NO:151) AE364 Ac-LRMKYP Cit-NH₂0.56 (SEQ ID NO:152) AE365 Ac-LR Harg MYPK-NH₂ 0.65 (SEQ ID NO:153)AE366 Ac-LR Harg KYP Cit-NH₂ 0.53 (SEQ ID NO:154) AE367 Ac-LRMMYPCit-NH₂ 0.69 (SEQ ID NO:155) AE368 Ac-LRLKYPN-NH₂ 0.55 (SEQ ID NO:156)AE301 Ac-LRLKYPK-NH₂ 0.54 (SEQ ID NO:127) AE370 Ac-LRMKYPN-NH₂ 0.55 (SEQID NO:157) AE371 Ac-FK Harg MYP Cit-NH₂ 0.61 (SEQ ID NO:158) AE114Ac-LRMKLPK-NH₂ 0.40 (SEQ ID NO:23) AE132 Ac-Y Harg MKLPKSAK-NH₂ 0.48(SEQ ID NO:48) AE134 Ac-YKMKLPKSAK-NH₂ 0.50 (SEQ ID NO:50) AE206Ac-LRLKLPK-NH₂ 0.46 (SEQ ID NO:70) AE197 Ac-LR Harg KLPK-NH₂ 0.45 (SEQID NO:61) AE220 Ac-LRMMLPK-NH₂ 0.63 (SEQ ID NO:81) AE241 Ac-LRMKLPCit-NH₂ 0.63 (SEQ ID NO:96) AE246 Ac-LRMKLPN-NH₂ 0.72 (SEQ ID NO:101)AE309 Ac-LRMKWPK-NH₂ 0.55 (SEQ ID NO:135)

Table 32: Activities of multiple substitution analogs of AE114,targeting the E^(k) allele, in a processed antigen assay. In this assay(as described in the legend of Table 30), the concentration of nativeHEL used during the prepulse was 20 μM. The concentration of AE peptideused was 64 μM for this assay. The supernatant dilution used in the HT-2cell assay was 1:4.

The results in this assay constitute, relatively, a mirror image of theresult in the simultaneous assay (Example 1, Table 16) and parallelthose of the antigenic peptide prepulse assay (Example 5, Table 26).

TABLE 33 Position 5 substitution analogs of AE114. Peptide SequenceE^(d) None 1.0 AE114 Ac-LRMKLPK-NH₂ 0.37 (SEQ ID NO:23) AE235Ac-LRMKYPK-NH₂ 0.45 (SEQ ID NO:92) AE322 Ac-LRMK (X1) PK-NH₂ 0.41 (SEQID NO:159) AE323 Ac-LRMK (X3) PK-NH₂ 0.25 (SEQ ID NO:159) AE324 Ac-LRMK(X4) PK-NH₂ 0.42 (SEQ ID NO:159) AE325 Ac-LRMK (X5) PK-NH₂ 0.33 (SEQ IDNO:159) AE326 Ac-LRMK (X6) PK-NH₂ 0.38 (SEQ ID NO:159) AE327 Ac-LRMK(X8) PK-NH₂ 0.60 (SEQ ID NO:159) AE328 Ac-LRMK (X9) PK-NH₂ 0.66 (SEQ IDNO:159) AE329 Ac-LRMK (X12) PK-NH₂ 0.32 (SEQ ID NO:159) AE330 Ac-LRMK(X13) PK-NH₂ 0.27 (SEQ ID NO:159) AE331 Ac-LRMK (X14) PK-NH₂ 0.50 (SEQID NO:159) AE332 Ac-LRMK (X15) PK-NH₂ 0.48 (SEQ ID NO:159)

Table 33: Activities of position 5 substitution analogs of AE114 in aprocessed antigen assay. In this assay (as described in the legend ofTable 30) the concentration of native HEL used during the prepulse was10 μM. The concentration of AE peptide used was 64 μM for this assay.The supernatant dilution used in the HT-2 cell assay was 1:8. Thefollowing side chain structures were substituted at position 5:X2=p-chloro-Phe; X3=p-fluoro-Phe; X4=p-nitro-Phe;X5=α-amino-4-phenylbutyrate; X6=β-thienylalanine (Thi);X8=di-bromo-tyrosine; X9=di-iodo-tyrosine; X12=β-1-napthyl-alanine;X13=β-2-napthyl-alanine; X14=1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid (Tic); andX15=1,2,3,4-tetrahydroisoquinoline-7-hydroxy-3-carboxylic acid[Tic(OH)].

The results in this assay constitute, relatively, a mirror image of theresult in the simultaneous assay (Example 1, Table 17 and parallel thoseof the antigenic peptide prepulse assay (Example 5, Table 27).

TABLE 34 Cyclical analogs of AE-114. Peptide Sequence E^(d) None 1.0AE114 Ac-LRMKLPK-NH₂ 0.22 (SEQ ID NO:23) AE381 Ac-LRMKLPK-NH₂ 0.18 (SEQID NO:23) AE382 Ac-LRMKLPK-NH₂ 0.61 (SEQ ID NO:23) AE235 Ac-LRMKYPK-NH₂0.19 (SEQ ID NO:92)

Table 34: Activities of cyclical analogs of AE114 in a processed antigenassay. In this assay (as described in the legend of Table 30) theconcentration of AE peptide used was 64 μM for this assay. Thesupernatant dilution used in the HT-2 cell assay was 1:8. The AE381peptide is a head-to-tail cyclization: the amino terminal amino group iscoupled through an amide linkage to the carboxyl terminal group. The AE382 peptide is a side-to-tail cyclization: the epsilon amino group ofLys⁴ is coupled through an amide linkage to the carboxyl terminal group.The concentration of native HEL used during the prepulse was 10 μM.

The results in this assay constitute, relatively, a mirror image of theresult in the simultaneous assay (Example 1, Table 18) and parallelthose of the antigenic peptide prepulse assay (Example 5, Table 2B).

TABLE 35 Activities of multiple substitution analogs of AE235 in anAE101 series peptide prepulse assay. Peptide Sequence E^(k) E^(d) NONE1.0 1.0 AE381 LRMKLPK 0.3 1.0 (SEQ ID NO:23) AE382 LRMKLPK 3.2 1.7 (SEQID NO:23) AE114 Ac-LRMKLPK-NH₂ 2.0 6.0 (SEQ ID NO:23) AE235Ac-LRMKYPK-NH₂ 2.1 15.6 (SEQ ID NO:92) AE206 Ac-LRLKLPK-NH₂ 2.7 5.0 (SEQID NO:70) AE117 Ac-YRMK-NH₂ 2.3 4.4 (SEQ ID NO:26) AE366 Ac-LR Harg KYPCit-NH₂ 3.5 7.5 (SEQ ID NO:154) AE172 Ac-LRNKlpk-NH₂ 2.1 1.1 (SEQ IDNO:123) AE230 Ac-LRMKDPK-NH₂ 1.6 0.8 (SEQ ID NO:87) AE331 Ac-LRMK (X14)PK-NH₂ 1.2 1.3 (SEQ ID NO:159) AE332 Ac-LRMK (X15) PK-NH₂ 2.5 1.7 (SEQID NO:159)

Table 35: Activities of multiple substitution analogs of AE235,targeting the E^(k) and E^(d) allele, in an AE101 series peptideprepulse assay. In this assay the concentration of AE101 series peptidesused during the prepulse was 64 μM. The concentrations of antigenicpeptides were 0.3 μM for Ed and 0.4 μM for E^(k). The supernatantdilutions taken into the HT-2 cell assay was 1:4 for E and 1:2 forE^(k). The AE381 peptide is a head-to-tail cyclization: the aminoterminal amino group is coupled through an amide linkage to the carboxylterminal group. The AE 382 peptide is a side-to-tail cyclization: theepsilon amino group of Lys⁴ is coupled through an amide linkage to thecarboxyl terminal group.

AE101 series peptide prepulse assays were carried out in the E^(d) andE^(k) systems as described for simultaneous competition assays with thefollowing modifications. Fixed APC were first incubated for 6 h at1×10⁶DMEM-5% FCS with 64 μM of each AE101 series peptide or with PBS.The APC were then washed four times with ten volumes of DMEM-5% FCS, andwere cocultured with T hybridoma cells and the indicated submaximaldoses of antigenic peptides used in the simultaneous competition assays.The baseline T cell response was measured by culturing T hybridoma cellswith antigenic peptide and PBS-pretreated APC.

The results of this assay demonstrate that the low activity of AE381(cyclic LRMKLPK) (SEQ ID NO: 23) in the simultaneous assay (Example 1,Table 18) and its potent activities in the antigenic peptide prepulseassay (Example 5, Table 28) and in the processed antigen assay (Example6, Table 34) are paralleled by its potent suppressive activity in theAE101 series peptide prepulse assay. These sets of data support the viewthat AE381 binds tightly to the allosteric effector site, withoutallowing for the substitution of the antigenic peptide at the antigenicpeptide binding site by a second antigenic peptide.

Example 7 Mechanisms of AE101 Series Compound-Induced Release of HumanMyelin Basic Protein (hMBP) Peptide (90-102) From the Antigenic PeptideBinding Site of HLA-DR1 Molecules

Four assays with purified HLA-DR1 molecules were established todetermine the molecular mechanisms of AE101 series compounds withrespect to binding, release, or exchange of antigenic peptides at theirbinding site in MHC class II molecules. These experiments defineparameters for the most effective therapeutic use of individual AE101series compounds.

The four assays measure the following. (I) The effect of AE101 seriescompounds to release a biotinylated antigenic peptide from the antigenicpeptide binding site, without the concerted effect of a second,unlabeled antigenic peptide to promote release of the biotinylatedantigenic peptide. (II) The effect of AE101 series compounds to releasea biotinylated antigenic peptide from the antigenic peptide bindingsite, with a second, unlabeled antigenic peptide promoting release ofthe biotinylated antigenic peptide. (III) The effect of AE101 seriescompounds to promote the binding of a biotinylated antigenic peptideinto MHC class II molecules which have already been loaded with asecond, antigenic peptide. (IV) The effect of AE101 series compounds topromote the binding of a biotinylated antigenic peptide into anantigenic peptide binding site of MHC class II molecules which have notbeen loaded with a second, antigenic peptide. With respect to these lasttwo assays, it is relevant to note that the MHC class II molecules whichwere prepared for these assays were synthesized in cultured insect cellswhich were infected with an insect virus carrying the genes for thehuman MHC class II molecules. These human MHC class II proteins areproduced in the cultured insect cells without any peptides occupying theantigenic peptide binding site (Stern, L. J., and Wiley, D. C., Cell 68:465-477, (1992)).

These assays were performed with a soluble form of the MHC class IImolecule which is truncated as an exomembranal construct. A peptidesequence was added to the C-terminus of the MHC class II alpha chain, sothat the heterodimeric MHC class II alpha, beta chain complex could betethered in the assay well by a monoclonal antibody to that peptidyl“tail” without apparently affecting the conformational changes in MHCclass II molecules induced by AE101 compounds. Elimination of thehydrophobic, transmembranal segments of each chain of the receptorreduces substantial background binding of certain assay components.Putting the “tail” on the alpha chain rather than on the beta chain ispreferred because little genetic polymorphism of the alpha chain isfound in humans, while there is great polymorphism of the beta chain.Inherited susceptibility to certain autoimmune diseases is linked tosome beta chain forms. In future studies of drug design to control suchdiseases on a MHC Class II allele-specific basis, it will be convenientto construct multiple assay complexes with the constant, “tailed”,truncated alpha chain and various truncated forms of beta chains withoutsuch tails. Designing this assay with the “tail” on only the alpha chainis thus a preferred characteristic.

Certain constructs of the genes for the HLA-DR alpha and beta chains ofthe MHC class II molecules were prepared. The HLA-DR alpha gene wastruncated after Asn¹⁹² to remove transmembranal and cytoplasmic regionsfrom the protein product. Codons for a nine amino acid HA epitope (SEQID NO: 167) were attached to the gene for the alpha chain after thecodon for Asn¹⁹² to permit binding of the modified protein product by amonoclonal antibody 12CA5 (Kolodziej, P. A., and Young, R. A., MethodsEnzymol. 194: 508-519 (1991)). A gene coding for the HLA-DR1 beta chainwas truncated after position Lys¹⁹⁸, again to delete the transmembranalregion. Each gene was cloned into a respective Baculovirus (BV) bystandard molecular biological methods. The insect cell line HS(Invitrogen) was co-infected with the two, respective, purifiedBV-HLA-DR alpha and BV-HLA-DR1 beta clones. Three days afterco-infection, the supernatant of the HS cell culture was brought to 1 mMiodoacetamide, 1 mM phenylmethylsulfonyl fluoride, and 1 mMethylenediaminetetraacetic acid (EDTA), and was collected andconcentrated 10-15 times by ultrafiltration over an Amicon YM30membrane.

The general form of the biotinylated antigenic peptide release assay wasperformed as follows. The wells of a 96-well microtiter plate werecoated with the 12CA5 anti-HA tail antibody (2.5 μg/mL in 50 mM sodiumcarbonate, pH 9.6) at 4° C. overnight Those coated microtiter wells werethen blocked with bovine gelatin (2 mg/mL) for 3-5 h at 4° C.Concentrated H5 cell culture supernatant (100 μl) was added to each welland the plates were incubated at 4° C. for 2 h. N-terminallybiotin-labeled hMBP(90-102) (50 μM/75 μl) in PBS with 0.02% sodium azideand 1 mM EDTA was added to each well and the plates were incubated at37° C. overnight. After washing with 0.05% Tween in PBS (100 μl), AE100series compounds in PBS with 0.02% sodium azide and 1 mM EDTA were addedat indicated concentrations and the plates were incubated at 37° C. for1 h. After washing, avidin-conjugated horseradish peroxidase (HRP) wasadded and incubated at 4° C. for 1 h. After washing, 100 μl of HRPsubstrate 3,3′,5,5′, -tetramethyl-benzidine (Sigma) was added to eachwell and the plates were incubated at room temperature for 5 min. 25 μlof 2 N sulfuric acid was added to each well to stop the reaction. Thecolorimetric change at 450 nm was quantitated with an ELISA reader(Molecular Devices). Each value was the average of duplicates and eachobservations was made at least twice. Comparable results were obtainedin initial experiments with biotin-labeled influenza virus hemagglutininpeptide (307-319) (PKYVKQNTLKLAT) (SEQ ID NO:164), and with influenzavirus matrix peptide (18-29) (GPLKAEIAQRLE) (SEQ ID NO:165) both ofwhich have also been shown to bind to HLA-DR1. The specific variationsfor three additional assays are presented in the legend of each Table.

I. Induction of release of hMBP(90-102) from HLA-DR1 by some AE101series compounds.

In Table 36, it is apparent that some AE101 compounds completely releasebiotin-labeled hMBP(90-102) from DR1 molecules. Some other compounds,which were very effective in murine in vitro assays for antigenpresentation, did not effectively release biotinylated hMEP(90-102). Forexample, AE235 peptide, which was effective in various murine MHC classII in vitro assays for the presentation of specific antigenic peptidesby antigen presenting cells to their respective T cell hybridomas, didnot release bound antigenic peptide hMBP(90-102) from HLA-DR1 while someother, longer AE101 series compounds did. The motif of residues in AE101series compounds required for release of biotinylated antigenic peptideswithout the presence of a second unlabeled antigenic peptide wasdetermined. Among a series of homologs, the N-terminal 12 amino acidpeptide AE107 but not the N-terminal 10 amino acid peptide AE108released biotin-labeled hMBP(90-102) from the HLA-DR1 molecules,indicating that AE108 did not contain amino acids which played a role inreleasing hMBP(90-102) from HLA-DR1 molecules. The N-terminal portion ofAE100, AE401, however, retained capacity to release the hMBP(90-102)peptide from HLA-DR1 molecules. In additional experiments, AE101, AE106,and AE107 at 125 nM completely release hMBP(90-102) from HLA-DR1 underthe conditions of this assay.

TABLE 36 Induction of release of hMBP (90-102) from HLA-DR1 by someAE101 series compounds. Peptide AE# Sequence Relative Release Pos.Control 1.00 Neg. Control 0.00 AE101 YRMKLPKSAKPVSQMR −0.33 (SEQ IDNO:3) AE106 YRMKLPKSAKPVSQ −0.12 (SEQ ID NO:14) AE107 YRMKLPKSAKPV −0.07(SEQ ID NO:15) AE108 YRMKLPKSAK 1.14 (SEQ ID NO:16) AE103 KLPKSAKPVSQMR0.01 (SEQ ID NO:11) AE104 PKSAKPVSQMR 0.02 (SEQ ID NO:12) AE105SAKPVSQMR 0.16 (SEQ ID NO:13) AE100 YRMKLPKPPKPVSKMR 0.37 (SEQ ID NO:2)AE401 Ac-LRMKLPKPP-NH₂ 0.16 (SEQ ID NO:160) AE402 Ac-LRMKLPKPPKPV-NH₂0.65 (SEQ ID NO:161) AE403 Ac-MKLPKPPKPV-NH₂ 0.64 (SEQ ID NO:162) AE405Ac-LPKSAKPV-NH₂ 0.11 (SEQ ID NO:163) AE235 Ac-LRMKYPK-NH₂ 0.79 (SEQ IDNO:92)

Table 36. Induction of release of hMBP(90-102) from HLA-DR1 by someAE101 series compounds. AE101 series compounds were tested at 64 μM. Thepositive control wells contained only PBS without an AE101 seriescompound. The positive control value was set at 1.0. The negativecontrol wells contained supernatant of H5 cells infected with wild typeBaculovirus and then biotin-labeled hMBP was added. The negative controlvalue was set at 0.0. Relative release of biotinylated antigenic peptidewas expressed as the fraction the o.d. of the experimental value was ofthe o.d. of the positive control value.

II. The release of antigenic peptides from HLA-DR1 complexes iscatalyzed by certain AE101 series compounds only in the presence ofunlabeled antigenic peptide.

While some AE compounds effectively release bound biotin-labeledhMBP(90-102), some other AE101 series compounds can not. Those laterAE101 series compounds were then tested for release of hMBP(90-120) inthe presence of excess unlabeled hMBP. In the presence of excessunlabeled hMBP(90-120), some AE101 series compounds, which do notrelease antigenic peptide from DR molecules in the absence of excessunlabeled hMBP(90-120), can effectively release bound hMBP effectivelyin the presence of excess unlabeled hMBP(90-120)

TABLE 37 Release of antigenic peptides from HLA-DR1 com- plexescatalyzed by certain AE101 series compounds only in the presence ofunlabeled antigenic pep- tide. Relative Release Without With unlabeledunlabeled Pep- hMBP(90-102) hMBP(90-102) tide Sequence 1 μM 64 μM 1 μM64 μM AE# AE381 cyclic LRMKLPK 0.64 0.5 0.09 −0.19 (SEQ ID NO:23) AE206Ac-LRLKLPK-NH₂ 0.58 0.08 0.03 −0.29 (SEQ ID NO:70) AE108 YRMKLPKSAK 0.520.31 0.00 0.14 (SEQ ID NO:16) AE143 Ac-YRMKLhydrpKSAK-NH₂ 0.51 0.58 0.350.21 (SEQ ID NO:94) AE235 AC-LRMKYPK-NH₂ 1.05 0.81 0.48 0.6 (SEQ IDNO:92)

Table 37. Some AE101 series compounds catalyze the release ofbiotin-labeled hMBP(90-102) from HLA-DR1 molecules only in the presenceof unlabeled hMBP. AE101 series compounds were tested at 1 μM and at 64μM. The experimental procedures were the same as reported for Table M1,except that unlabeled hMBP(90-102) (250 μM) was added in some wellsduring the release-inducing step, as indicated. Release ofbiotin-labeled hMBP(90-102) by unlabeled hMBP(90-102) alone withoutAE101 series compounds was 0.81, and release of biotin-labeledhMBP(90-102) by AE101 at 1 μM without hMBP(90-102) was 0.02. Thepresence of unlabeled antigenic peptide in the solution greatly enhancedthe release of biotinylated antigenic peptide by certain AE101 seriescompounds.

III. Certain AE101 series compounds exchange biotinylated antigenicpeptides into antigenic peptide-loaded HLA-DR molecules.

Some AE101 compounds were found to release bound antigenic peptide fromMHC class II molecules in the presence of excess unlabeled antigenicpeptide. Next, the activity of AE101 series compounds to promote theexchange of the antigenic peptide with respect to MHC class II moleculeswas tested. Certain AE101 series compounds promote the exchange ofantigenic peptides with respect to MHC class II molecules. AE101 seriescompounds with this activity usually also had the ability to releaseantigenic peptides from the MHC class II molecules.

TABLE 38 Certain AE101 series compounds exchange hMBP (90- 102) intoHLA-DR1 molecules. Peptide AE# Sequence Relative Binding Pos. Control1.00 Neg. Control 0.00 No enhancement 0.08 AE101 YRMKLPKSAKPVSQMR 0.31(SEQ ID NO:3) AE107 YRMKLPKSAKPV 1.04 (SEQ ID NO:15) AE381 cyclicLRMKLPK −0.12 (SEQ ID NO:23) AE206 Ac-LRLKLPK-NH₂ −0.13 (SEQ ID NO:70)AE100 YRMKLPKPPKPVSKMR 0.31 (SEQ ID NO:2) AE401 Ac-LRMKLPKPP-NH₂ 0.45(SEQ ID NO:160) AE402 Ac-LRMKLPKPPKPV-NH₂ −0.03 (SEQ ID NO:161) AE403Ac-MKLPKPPKPV-NH₂ 0.40 (SEQ ID NO:162) AE405 Ac-LPKSAKPV-NH₂ 0.57 (SEQID NO:163) AE235 Ac-LRMKYPK-NH₂ 0.15 (SEQ ID NO:92)

Table 38. Certain AE101 series compounds exchange biotinylatedhMBP(90-102) into antigenic peptide-loaded HLA-DR1 molecules. Theexperimental procedures were the same as described in Table M1 exceptthat unlabeled hMBP(90-102) (50 μM) was first incubated with purified,soluble HLA-DR1 molecules overnight, and the cells were then washed. Theexchange step was then performed with AE101 series compounds in thepresence of biotin-labeled hMBP(90-102) (50 μM) at 37° C. for 1 h. Thepositive control was the HLA-DR molecules incubated with biotin-labeledhMBP(90-102) (50 MM) overnight. The negative control was wild typesupernatant incubated with biotin-labeled hMBP(90-102). “No enhancement”refers to the performance of the exchange step in the presence ofbiotin-labeled hMBP(90-102) (50 μM) without AE101 series compounds at37° C. for 1 h. Relative binding was the fraction the o.d. of theexperimental value was of the o.d. of the positive control value.

IV. Certain AE compounds promote the binding of antigenic peptide to“empty” HLA-DR molecules.

This experiment addressed whether AE101 series compounds might induce aconformational change in the nascent, “empty” MHC class II molecules tofacilitate binding of antigenic peptide to MHC class II molecules.Insect cell-produced HLA-DR1 molecules are known to be empty (Stern, L.J., and Wiley, D. C., Cell 68:465-477, 1992). Some AE101 seriescompounds promoted the binding of hMBP to HLA-DR1 molecules. Thisfinding indicated that AE101 series compounds induced a conformationalchange favoring the binding of hMBP.

TABLE 39 AE101 Series compounds promote the binding of hMBP (90-102) to“empty” HLA-DR1 mole- cules. Relative Peptide AE# Sequence EnhancementNo enhancement 1 Neg. control 0.00 AE101 YRMKLPKSAKPVSQMR 2.66 (SEQ IDNO:3) AE107 YRMKLPKSAKPV 2.59 (SEQ ID NO:15) AE381 cyclic LRMKLPK 1.84(SEQ ID NO:23) AE206 Ac-LRLKLPK-NH₂ 2.57 (SEQ ID NO:70) AE100YRMKLPKPPKPVSKMR 3.44 (SEQ ID NO:2) AE401 Ac-LRMKLPKPP-NH₂ 4.29 (SEQ IDNO:160) AE402 Ac-LRMKLPKPPKPV-NH₂ 0.33 (SEQ ID NO:161) AE403Ac-MKLPKPPKPV-NH₂ 0.47 (SEQ ID NO:162) AE405 Ac-LPKSAKPV-NH₂ 0.43 (SEQID NO:163) AE235 Ac-YRMKYPK-NH₂ 0.61 (SEQ ID NO:92)

Table 39. Certain AE101 series compounds promote the binding ofbiotin-labeled hMBP(90-102) to “empty” HLA-DR1 molecules. After HLA-DR1molecules were immobilized onto the plate, the wells were washed 3 timeswith 0.05% Tween in PBS. They were then incubated biotin-labeledhMBP(90-102) (50 μM) in PBS with 1 mM EDTA and the indicated AE101series compound (64 μM) at 37° C. for 1 h. The wells were washed anddeveloped with avidin conjugated HRP, followed by a colorimetric assay.Relative enhancement was the fraction the o.d. of the experimental valuewas of the o.d. of the positive control value. No enhancement was thevalue seen with the HLA-DR1 molecules incubated with biotin-labeledhMBP(90-102) without the AE101 series compound at 37° C. for 1 h and wasset at 1.0. The negative control was with supernatant of a culture withwild type Baculovirus with biotin-labeled hMBP(90-102) at 37° C. for 1h. That value was set at 0.0.

Data of this Example reveal three classes or groups of AE101 seriescompounds which can be identified in terms of their differing activitiesto release, to exchange and/or to promote the binding of antigenicpeptides with respect to HLA-DR1 molecules. These three activityclasses, which also relate to the structures of the compounds, lead to amolecular mechanism model which is consistent with two x-raycrystallographic studies of peptide binding into the antigenic peptidebinding site of MHC class II molecules. The three, empirical patterns ofactivity are the following. 1) Group One. Certain AE101 series compoundsefficiently release bound antigenic peptide from HLA-DR1 molecules inthe absence of additional antigenic peptide. They also efficientlyreplace bound antigenic peptide with a second unbound antigenic peptide.Since these compounds also promote the initial binding of antigenicpeptide to freshly prepared, “empty” DR1 molecules, as synthesized inthe insect virus/cell line system, they appear to induce aconformational change in such DR1 molecules to promote or permit initialbinding of antigenic peptides. 2) Group Two. Other AE101 seriescompounds cannot efficiently release bound antigenic peptide fromHLA-DR1 molecules in the absence of unlabeled antigenic peptide, andrelease bound antigenic peptide but only in the presence of excessunlabeled antigenic peptide. This subset of AE101 series compounds doesnot efficiently promote the binding of antigenic peptides to nascentHLA-DR1 molecules. 3) Group Three. Yet other AE101 series compoundsdemonstrate little activity in releasing, exchanging, or promoting thebinding of antigenic peptides to HLA-DR1 molecules.

Assignment of individual AE101 series compounds to each of these threeclasses might vary depending upon the MHC class II alleles and thespecies being studied in a given screening assay. In the studies of thisdisclosure, various AE101 series compounds demonstrate significantdegrees of allele and species specificity.

The data of this disclosure indicate varying molecular mechanisms bywhich AE101 series compounds release or promote the binding of antigenicpeptides to MHC class II molecules. These mechanisms can be interpretedin terms of ways in which AE101 series compounds might bind to adifferent binding site than where antigenic peptides bind to MHC classII molecules, that is, to an allosteric site. The binding of AE101series compounds to such an allosteric regulatory site appears to loosenthe antigenic peptide binding site to release antigenic peptide and topromote the binding of antigenic peptide. From the functional data aloneone might propose that Group One AE101 series compounds might open theantigenic peptide completely so that the MHC class II molecules canrelease, exchange, and promote the charging of second antigenicpeptides. Group Two AE101 series compounds appear only partially to openthe antigenic peptide binding site. Such a limited action loosens thebound antigenic peptide but an additional force, such as that induced bythe binding of a second antigenic peptide, is required for thesubstitution of the first antigenic peptide.

These hypothesized molecular mechanisms can be related to certainstructural specifications for each of the AE101 series compounds. Thesemechanisms also relate to the crystallographic images of certainpeptides bound to MHC class II molecules, containing either antigenicpeptide or the Ii protein-derived “CLIP” peptide (Stern et al., Nature368: 215-221 (1994) and Ghosh et al., Nature 378: 457-462 (1995)).First, it was observed that certain AE101 series compounds comprisinghomologs of up to the 10 N-terminal amino acids of the AE101 peptidecatalyze the release of biotinylated antigenic peptide from theantigenic peptide binding site only in the presence in solution ofadditional antigenic peptide. Without the presence of that additionalantigenic peptide, these 10 amino acid or less homologs of AE101presumably only bind to the MHC class II molecule. Secondly, it wasobserved that AE101 series compounds comprising homologs of 12 or moreof the amino acids from the N-terminus of AE101, or better yet runningfrom positions Leu⁵ to Val¹² in AE101, induce the dissociation of boundantigenic peptide from the antigenic peptide binding site of MHC classII molecules without the requirement for additional antigenic peptidebeing present in the solution. The motif required for this autocatalyticeffect on release of antigenic peptides, thus, did not extend to theN-terminus of the AE101 peptide since the residues of AE405, comprisingresidue positions 5 through 12 of AE101, were sufficient to releaseantigenic peptide without the presence of excess quantities in solutionof a second antigenic peptide. In light of the crystallographic data ofStern et al. and Ghosh et al., one can propose that, if the Ii proteinlies in MHC class II molecules in registry with the positioning of theCLIP peptide of Ii within the antigenic peptide binding site and theAE101 series compounds lie in registry with that hypothesizedpositioning of the Ii protein, then the following subsites can beidentified. First, there is the antigenic peptide binding troughextending C-terminally from p⁸⁵ in Ii (p⁶ in AE101 peptide). Secondly,there is a core of the AE101 structure represented by AE114 which cancatalyze the release of antigenic peptide from the antigenic peptidebinding site only in the presence of excess quantities of a secondantigenic peptide. Thirdly, there is a subsite ranging from p⁵ throughv¹² (partially overlapping the second allosteric effector site) which issufficient to exchange antigenic peptide from the antigenic peptidebinding site.

167 1 16 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 1 Leu Arg Met Lys Leu Pro Lys Pro Pro Lys Pro Val SerLys Met Arg 1 5 10 15 2 16 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 2 Tyr Arg Met Lys Leu Pro Lys ProPro Lys Pro Val Ser Lys Met Arg 1 5 10 15 3 16 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 3 Tyr Arg Met LysLeu Pro Lys Ser Ala Lys Pro Val Ser Gln Met Arg 1 5 10 15 4 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide4 Leu Arg Met Lys Leu Pro Lys 1 5 5 15 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 5 Ala Met Lys ArgHis Gly Leu Asp Asn Tyr Arg Gly Tyr Ser Leu 1 5 10 15 6 16 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide6 Asn Thr Asp Gly Ser Thr Asp Tyr Gly Ile Leu Gln Ile Asn Ser Arg 1 5 1015 7 11 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 7 Asn Ala Trp Val Ala Trp Arg Asn Arg Cys Lys 1 5 10 824 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 8 Ile Phe Ala Gly Ile Lys Lys Lys Ala Glu Arg Ala Asp Leu IleAla 1 5 10 15 Tyr Leu Lys Gln Ala Thr Ala Lys 20 9 22 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 9 Phe AlaGly Leu Lys Lys Ala Asn Glu Arg Ala Asp Leu Ile Ala Tyr 1 5 10 15 LeuLys Gln Ala Thr Lys 20 10 15 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 10 Arg Met Lys Leu Pro Lys Ser AlaLys Pro Val Ser Gln Met Arg 1 5 10 15 11 13 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 11 Lys Leu Pro LysSer Ala Lys Pro Val Ser Gln Met Arg 1 5 10 12 11 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 12 Pro Lys Ser AlaLys Pro Val Ser Gln Met Arg 1 5 10 13 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 13 Ser Ala Lys ProVal Ser Gln Met Arg 1 5 14 14 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 14 Tyr Arg Met Lys Leu Pro Lys SerAla Lys Pro Val Ser Gln 1 5 10 15 12 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 15 Tyr Arg Met Lys Leu Pro LysSer Ala Lys Pro Val 1 5 10 16 10 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 16 Tyr Arg Met Lys Leu Pro Lys SerAla Lys 1 5 10 17 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 17 Leu Arg Met Lys Leu Pro Lys Ser Ala Lys 15 10 18 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 18 Leu Arg Met Lys Leu Pro Lys Pro Pro Pro 1 5 10 1910 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 19 Leu Arg Met Lys Leu Pro Lys Pro Pro Lys 1 5 10 20 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide20 Tyr Arg Met Lys Leu Pro Lys Ser Ala 1 5 21 8 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 21 Tyr Arg Met LysLeu Pro Lys Ser 1 5 22 7 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 22 Tyr Arg Met Lys Leu Pro Lys 1 523 7 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 23 Leu Arg Met Lys Leu Pro Lys 1 5 24 6 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 24 Tyr ArgMet Lys Leu Pro 1 5 25 5 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 25 Tyr Arg Met Lys Leu 1 5 26 4PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 26 Tyr Arg Met Lys 1 27 3 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 27 Tyr Arg Met 1 28 11 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide28 Ser Leu Arg Met Lys Leu Pro Lys Ser Ala Lys 1 5 10 29 12 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide29 Asp Ser Leu Arg Met Lys Leu Pro Lys Ser Ala Lys 1 5 10 30 13 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide30 Leu Asp Ser Leu Arg Met Lys Leu Pro Lys Ser Ala Lys 1 5 10 31 14 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide31 Gln Leu Asp Ser Leu Arg Met Lys Leu Pro Lys Ser Ala Lys 1 5 10 32 15PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 32 Leu Gln Leu Asp Ser Leu Arg Met Lys Leu Pro Lys Ser Ala Lys 15 10 15 33 16 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 33 Asn Leu Gln Leu Asp Ser Leu Arg Met Lys Leu Pro LysSer Ala Lys 1 5 10 15 34 7 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 34 Xaa Arg Met Lys Leu Pro Lys 1 535 7 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 35 Xaa Arg Met Lys Leu Pro Lys 1 5 36 7 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 36 Xaa ArgMet Lys Leu Pro Lys 1 5 37 7 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 37 His Arg Met Lys Leu Pro Lys 1 538 7 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 38 Lys Arg Met Lys Leu Pro Lys 1 5 39 7 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 39 Asp ArgMet Lys Leu Pro Lys 1 5 40 7 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 40 Glu Arg Met Lys Leu Pro Lys 1 541 7 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 41 Asn Arg Met Lys Leu Pro Lys 1 5 42 7 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 42 Gln ArgMet Lys Leu Pro Lys 1 5 43 7 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 43 Phe Arg Met Lys Leu Pro Lys 1 544 7 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 44 Met Arg Met Lys Leu Pro Lys 1 5 45 10 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide45 Tyr Ala Met Lys Leu Pro Lys Ser Ala Lys 1 5 10 46 11 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 46 Tyr ArgAsn Met Lys Leu Pro Lys Ser Ala Lys 1 5 10 47 10 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 47 Tyr Xaa Met LysLeu Pro Lys Ser Ala Lys 1 5 10 48 9 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 48 Tyr Xaa Met Lys Leu Pro LysSer Ala 1 5 49 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 49 His Met Lys Leu Pro Lys Ser Ala Lys 1 5 5010 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 50 Tyr Lys Met Lys Leu Pro Lys Ser Ala Lys 1 5 10 51 10 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide51 Tyr Asp Met Lys Leu Pro Lys Ser Ala Lys 1 5 10 52 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 52 Tyr GluMet Lys Leu Pro Lys Ser Ala Lys 1 5 10 53 10 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 53 Tyr Asn Met LysLeu Pro Lys Ser Ala Lys 1 5 10 54 10 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 54 Tyr Gln Met Lys Leu Pro LysSer Ala Lys 1 5 10 55 10 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 55 Tyr Phe Met Lys Leu Pro Lys SerAla Lys 1 5 10 56 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 56 Tyr Tyr Met Lys Leu Pro Lys Ser Ala Lys 15 10 57 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 57 Tyr Met Met Lys Leu Pro Lys Ser Ala Lys 1 5 10 5810 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 58 Tyr Leu Met Lys Leu Pro Lys Ser Ala Lys 1 5 10 59 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide59 Leu Arg Xaa Lys Leu Pro Lys 1 5 60 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 60 Leu Arg Xaa LysLeu Pro Lys 1 5 61 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 61 Leu Arg Xaa Lys Leu Pro Lys 1 5 62 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide62 Leu Arg His Lys Leu Pro Lys 1 5 63 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 63 Leu Arg Lys LysLeu Pro Lys 1 5 64 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 64 Leu Arg Asp Lys Leu Pro Lys 1 5 65 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide65 Leu Arg Glu Lys Leu Pro Lys 1 5 66 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 66 Leu Arg Asn LysLeu Pro Lys 1 5 67 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 67 Leu Arg Gln Lys Leu Pro Lys 1 5 68 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide68 Leu Arg Phe Lys Leu Pro Lys 1 5 69 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 69 Leu Arg Tyr LysLeu Pro Lys 1 5 70 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 70 Leu Arg Leu Lys Leu Pro Lys 1 5 71 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide71 Leu Arg Met Xaa Leu Pro Lys 1 5 72 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 72 Leu Arg Met XaaLeu Pro Lys 1 5 73 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 73 Leu Arg Met Xaa Leu Pro Lys 1 5 74 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide74 Leu Arg Met His Leu Pro Lys 1 5 75 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 75 Leu Arg Met AspLeu Pro Lys 1 5 76 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 76 Leu Arg Met Asp Leu Pro Lys 1 5 77 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide77 Leu Arg Met Asn Leu Pro Lys 1 5 78 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 78 Leu Arg Met GlnLeu Pro Lys 1 5 79 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 79 Leu Arg Met Phe Leu Pro Lys 1 5 80 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide80 Leu Arg Met Tyr Leu Pro Lys 1 5 81 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 81 Leu Arg Met MetLeu Pro Lys 1 5 82 8 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 82 Leu Arg Met Lys Arg Asn Pro Lys 1 5 83 7PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 83 Leu Arg Met Lys Xaa Pro Lys 1 5 84 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 84 Leu Arg Met LysXaa Pro Lys 1 5 85 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 85 Leu Arg Met Lys His Pro Lys 1 5 86 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide86 Leu Arg Met Lys Lys Pro Lys 1 5 87 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 87 Leu Arg Met LysAsp Pro Lys 1 5 88 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 88 Leu Arg Met Lys Glu Pro Lys 1 5 89 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide89 Leu Arg Met Lys Asn Pro Lys 1 5 90 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 90 Leu Arg Met LysGln Pro Lys 1 5 91 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 91 Leu Arg Met Lys Phe Pro Lys 1 5 92 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide92 Leu Arg Met Lys Tyr Pro Lys 1 5 93 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 93 Leu Arg Met LysMet Pro Lys 1 5 94 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 94 Tyr Arg Met Lys Leu Xaa Lys Ser Ala Lys 15 10 95 8 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 95 Leu Arg Met Lys Leu Pro Arg Asn 1 5 96 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide96 Leu Arg Met Lys Leu Pro Xaa 1 5 97 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 97 Leu Arg Met LysLeu Pro Xaa 1 5 98 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 98 Leu Arg Met Lys Leu Pro His 1 5 99 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide99 Leu Arg Met Lys Leu Pro Asp 1 5 100 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 100 Leu Arg Met LysLeu Pro Glu 1 5 101 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 101 Leu Arg Met Lys Leu Pro Asn 1 5 102 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide102 Leu Arg Met Lys Leu Pro Gln 1 5 103 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 103 Leu Arg Met LysLeu Pro Phe 1 5 104 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 104 Leu Arg Met Lys Leu Pro Tyr 1 5 105 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide105 Leu Arg Met Lys Leu Pro Met 1 5 106 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 106 Leu Arg Met LysLeu Pro Leu 1 5 107 10 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 107 Ala Arg Met Lys Leu Pro Lys Ser Ala Lys 15 10 108 10 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 108 Tyr Ala Met Lys Leu Pro Lys Ser Ala Lys 1 5 10 10910 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 109 Tyr Arg Ala Lys Leu Pro Lys Ser Ala Lys 1 5 10 110 10 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide110 Tyr Arg Met Ala Leu Pro Lys Ser Ala Lys 1 5 10 111 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 111 TyrArg Met Lys Ala Pro Lys Ser Ala Lys 1 5 10 112 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 112 TyrArg Met Lys Leu Ala Lys Ser Ala Lys 1 5 10 113 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 113 TyrArg Met Lys Leu Pro Ala Ser Ala Lys 1 5 10 114 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 114 TyrArg Met Lys Leu Pro Lys Ala Ala Lys 1 5 10 115 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 115 TyrArg Met Lys Leu Pro Lys Ser Ala Ala 1 5 10 116 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 116 Xaa Arg Met LysLeu Pro Lys 1 5 117 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 117 Leu Xaa Met Lys Leu Pro Lys 1 5 118 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide118 Leu Arg Xaa Lys Leu Pro Lys 1 5 119 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 119 Leu Arg Met XaaLeu Pro Lys 1 5 120 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 120 Leu Arg Met Lys Xaa Pro Lys 1 5 121 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide121 Leu Arg Met Lys Leu Xaa Lys 1 5 122 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 122 Leu Arg Met LysLeu Pro Xaa 1 5 123 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 123 Lys Pro Leu Lys Xaa Xaa Xaa 1 5 124 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide124 Leu Arg Met Lys Leu Xaa Xaa 1 5 125 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 125 Xaa Arg Met LysLeu Pro Lys 1 5 126 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 126 Leu Arg Met Lys Xaa Pro Lys 1 5 127 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide127 Leu Arg Leu Lys Tyr Pro Lys 1 5 128 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 128 Leu Arg Xaa LysLeu Pro Lys 1 5 129 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 129 Leu Arg Xaa Lys Tyr Pro Lys 1 5 130 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide130 Leu Arg Xaa Lys Xaa Pro Lys 1 5 131 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 131 Leu Arg Xaa LysTyr Pro Xaa 1 5 132 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 132 Leu Arg Xaa Lys Xaa Pro Xaa 1 5 133 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide133 Leu Arg Leu Lys Tyr Pro Xaa 1 5 134 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 134 Leu Arg Leu LysXaa Pro Lys 1 5 135 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 135 Leu Arg Leu Lys Trp Pro Lys 1 5 136 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide136 Leu Arg Xaa Lys Xaa Pro Lys 1 5 137 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 137 Leu Arg Leu LysXaa Pro Lys 1 5 138 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 138 Leu Xaa Met Lys Asn Pro Lys 1 5 139 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide139 Leu Xaa Asn Lys Leu Pro Lys 1 5 140 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 140 Ala Arg Asn LysLeu Pro Lys 1 5 141 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 141 Ala Arg Met Lys Asn Pro Lys 1 5 142 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide142 Ala Arg Asn Lys Asn Pro Lys 1 5 143 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 143 Ala Arg Asn LysAsn Pro Phe 1 5 144 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 144 Leu Arg Asn Lys Asn Pro Phe 1 5 145 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide145 Leu Arg Asn Lys Asn Pro Lys 1 5 146 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 146 Leu Arg Met LysAsn Pro Phe 1 5 147 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 147 Ala Xaa Asn Lys Asn Pro Lys 1 5 148 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide148 Leu Xaa Met Lys Tyr Pro Lys 1 5 149 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 149 Leu Xaa Leu LysTyr Pro Lys 1 5 150 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 150 Leu Lys Met Lys Tyr Pro Lys 1 5 151 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide151 Leu Lys Xaa Lys Tyr Pro Lys 1 5 152 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 152 Leu Arg Met LysTyr Pro Xaa 1 5 153 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 153 Leu Arg Xaa Met Tyr Pro Lys 1 5 154 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide154 Leu Arg Xaa Lys Tyr Pro Xaa 1 5 155 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 155 Leu Arg Met MetTyr Pro Xaa 1 5 156 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 156 Leu Arg Leu Lys Tyr Pro Asn 1 5 157 7 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide157 Leu Arg Met Lys Tyr Pro Asn 1 5 158 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 158 Phe Lys Xaa MetTyr Pro Asn 1 5 159 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 159 Leu Arg Met Lys Xaa Pro Lys 1 5 160 9 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide160 Leu Arg Met Lys Leu Pro Lys Pro Pro 1 5 161 12 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 161 LeuArg Met Lys Leu Pro Lys Pro Pro Lys Pro Val 1 5 10 162 10 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 162 MetLys Leu Pro Lys Pro Pro Lys Pro Val 1 5 10 163 8 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 163 Leu Pro Lys SerAla Lys Pro Val 1 5 164 13 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 164 Pro Lys Tyr Val Lys Gln AsnThr Leu Lys Leu Ala Thr 1 5 10 165 12 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 165 Gly Pro Leu LysAla Glu Ile Ala Gln Arg Leu Glu 1 5 10 166 7 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 166 Lys Pro Leu LysMet Arg Leu 1 5 167 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic epitope 167 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5

What is claimed is:
 1. A method for inhibiting presentation of an MHCclass II-restricted antigenic peptide to a T cell, comprising: a)forming an incubation mixture comprising the following components underphysiological conditions: i) an MHC class II-expressing antigenpresenting cell displaying on its surface the MHC class II-restrictedantigenic peptide; ii) a peptide selected from the group of peptidesconsisting of SEQ ID NOS: 16, 18, 19, 22, 24, 25, and any one listed inTables 20-39; and b) contacting the incubated components of step a) witha T cell which is responsive to the MHC class II-restricted antigenicpeptide under physiological conditions wherein the peptide of ii)inhibits the presentation of the MHC class II-restricted antigenicpeptide to the T cell.